zebrafish research

Why Use Zebrafish to Study Human Diseases?

By Elizabeth Burke

Tuesday, August 9, 2016

Scientists use a variety of laboratory techniques to investigate the genetic cause of human diseases. Research often utilizes patients’ cells or tissue samples, but to determine if a mutation in a specific gene can cause a patient’s symptoms, we often need experimental animal models.

While mice and rats have been common choices for modeling human diseases in the past, the use of zebrafish is rapidly gaining popularity. Does this surprise you? Let me explain.

What are zebrafish?

Zebrafish are tropical fresh-water fish in the minnow family. In the wild, they are found in rivers and ponds of India, however they are now often available in pet shops. The name “zebrafish” comes from the horizontal blue stripes on each side of their bodies.

Zebrafish, so named due to their stripes, prefer to live in large groups called shoals.

How can you model a human disease in fish?

Although humans may appear to be extremely different than zebrafish, we are actually much more similar to them than you might think. In fact, 70% of human genes are found in zebrafish .

Moreover, zebrafish have two eyes, a mouth, brain, spinal cord, intestine, pancreas, liver, bile ducts, kidney, esophagus, heart, ear, nose, muscle, blood, bone, cartilage, and teeth. Many of the genes and critical pathways that are required to grow these features are highly conserved between humans and zebrafish. Thus, any type of disease that causes changes in these body parts in humans could theoretically be modeled in zebrafish.

Why use zebrafish when you could use mice?

While mice are evolutionarily more similar to humans because they are mammals, zebrafish have several advantages over their furry competitors.

One important advantage of zebrafish is that the adults are small and prefer to be housed in large groups, or “shoals”. As a result, they require much less space and are cheaper to maintain than mice.

The NIH Zebrafish Core houses hundreds of thousands of zebrafish in a state-of-the-art facility.

Another advantage is that adult zebrafish breed readily (approximately every 10 days) and can produce as many as 50 to 300 eggs at a time. This is quite different from mice as they generally produce litters of one to 10 pups and can only bear approximately three litters in their lifetime. Scientific experiments are generally repeated multiple times in order to prove that the results are accurate, so having an animal that can produce a large number of offspring over and over is helpful.

Zebrafish embryos are also laid and fertilized externally, which allows them to be easily manipulated in a variety of ways. In vitro fertilization can be performed if necessary. The one-cell-stage fertilized eggs can be easily injected with DNA or RNA to permanently modify their genetic makeup in order to generate transgenic or knock-out zebrafish lines. Working with mice in this way is much more complicated. Mouse embryos develop inside the mother, and to access and manipulate them the mother would have to be sacrificed. To keep the embryos alive after fertilizing or injecting them, they would need to be transplanted into another female mouse, as well.

Zebrafish larva, the stage of development from between three and thirty days post-fertilization, grow in length from approximately 3.5 to 8 millimeters.

Furthermore, zebrafish embryos are clear, which allows scientists to watch the fertilized eggs grow into fully formed baby fish under a microscope. Their transparency also enables the visualization of fluorescently labeled tissues in transgenic zebrafish embryos. Mouse embryos are not clear and develop inside the mother, so the observation of live embryo development like that in zebrafish is not possible.

However, there is a limit on what types of diseases can be studied in zebrafish. Human diseases caused by genes that do not exist in zebrafish require a different animal model. Additionally, zebrafish are not useful models for human diseases that mainly take place in a tissue type or body part that zebrafish do not have (e.g., prostate, mammary glands, lungs).

How exactly do you use zebrafish to investigate human diseases?

Often a patient’s DNA is sequenced in order to find a mutation in a gene that could potentially cause his or her disease symptoms. To determine if loss of function of that gene could cause the symptoms seen in the patient, the same gene is mutated or “knocked-out” in zebrafish, and then the fish are examined for similar symptoms. Although it is much more difficult to do, the exact mutation that the patient has can be introduced into zebrafish as well—this is called a “knock-in”.

If one or more of the patient’s symptoms are observed in the zebrafish knock-out or knock-in model, the zebrafish can be used for further studies to help determine why the mutation in that gene causes the disease. For instance, the structure of the muscle fibers can be examined for abnormalities under the microscope if the patient has a muscle disease. Or if the patient’s disease symptoms began during development in utero , knock-out or knock-in zebrafish embryos can be examined for gene expression changes (compared to embryos without the mutation) that could lead to abnormal development. For a patient with a neurological disease, the neurons of knock-out embryos can be fluorescently labeled to see if they form incorrectly.

In addition to utilizing zebrafish disease models to characterize human diseases, researchers can also identify and test new drugs to treat the diseases being modeled. The ability of zebrafish to generate many embryos every time they breed makes them especially useful for high throughput drug screening.

What are some examples of human diseases that have been successfully modeled in zebrafish?

The generation of a knock-out of the dystrophin gene in zebrafish has been shown to closely resemble the severity and progression of the human disease Duchenne muscular dystrophy. Patients with Duchenne muscular dystrophy have been found to carry mutations in dystrophin and demonstrate childhood muscle weakness that gets progressively worse. In both humans and the zebrafish model, the loss of dystrophin gradually leads to necrotic muscle fibers that are replaced by inflammatory cells, fibrosis, and abnormally sized muscle fibers.

This figure shows visual differences in muscle between wild-type zebrafish larva (A, B, C) and distrophic larva (A’, B’, C’). Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3484855/

Human melanoma has also been successfully modeled in zebrafish. The most commonly identified mutation in human melanomas—a single amino acid change in the gene BRAF —was created in zebrafish to make a knock-in model. Since cancers are caused by a combination of several genetic alterations, this knock-in zebrafish line was used to screen other potential cancer causing mutations. When another commonly observed melanoma mutation of the gene SETDB1 was added to the BRAF knock-in zebrafish, a melanoma rapidly developed. These results helped to establish that SETDB1 is an important gene in melanoma growth.

Images of a knock-in zebrafish that expresses the BRAF mutation alone (top) and one that was also injected with a transposon-based vector (miniCoopR) containing a mutant form of the gene SETDB1 (bottom). The addition of the SETB1 mutation resulted in melanoma (indicated by the arrow). Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3348545/

Those examples of how humans and zebrafish can manifest the same disease despite how different we appear make it is easy to understand why zebrafish are becoming a well-accepted animal model. Here in the NIH Undiagnosed Diseases Program, we perform studies using zebrafish as one of several approaches to investigate the potential involvement of altered genes in our patients’ extremely rare diseases. While mice have been the predominant animal bridge between the bench and bedside in the past, recent studies have demonstrated the potential of zebrafish to serve as a tractable alternative to mice. The timing of the adoption of zebrafish as an emerging model organism could not be better, as mouse studies often fail to translate to humans. Although no animal can perfectly model a human disease, I believe these little striped swimmers have great potential for advancing medical research in the future.

To learn more about how zebrafish contribute to biomedical science and human health, visit the websites for the Trans-NIH Zebrafish Initiative website and the NICHD Zebrafish Core .

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Study: Zebrafish are smarter than we thought

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A new study from MIT and Harvard University suggests that the brains of the seemingly simple zebrafish are more sophisticated than previously thought. The researchers found that larval zebrafish can use visual information to create three-dimensional maps of their physical surroundings — a feat that scientists didn’t think was possible.

In the new study, the researchers discovered that zebrafish can move around environmental barriers while escaping predators. The findings suggest that zebrafish are “much smarter than we thought,” and could be used as a model to explore many aspects of human visual perception, the researchers say.

“These results show you can study one of the most fundamental computational problems faced by animals, which is perceiving a 3D model of the environment, in larval zebrafish,” says Vikash Mansinghka, a principal research scientist in MIT’s Department of Brain and Cognitive Sciences and an author of the new study.

Andrew Bolton, an MIT research scientist and a research associate at Harvard University, is the senior author of the new study, which appears today in Current Biology . Hanna Zwaka, a Harvard postdoc, and Olivia McGinnis, a recent Harvard graduate who is now a graduate student at the Oxford University, are the paper’s lead authors.

Mapping the environment

Since the 1970s, zebrafish have been used to study a variety of human diseases, including cancer, cardiovascular disease, and diabetes. One of the early pioneers of zebrafish research was Nancy Hopkins, currently an MIT professor emerita of biology, who discovered many of the genes involved in zebrafish embryonic development.

More recently, scientists have begun to explore the possibility of using zebrafish as a model of behaviors that involve sensory perception. Three years ago, Bolton led a study showing that zebrafish can accurately predict the trajectories of their prey based on the prey’s position and velocity.

During that study, Bolton accidentally dropped one of the dishes containing larval zebrafish and noticed that the fish immediately scattered in all directions. That led him to wonder, was their choice of escape path totally random, and would it be affected if there were obstacles in the way?

The ability to detect obstacles requires integration of multiple types of sensory input, and the ability to use that information to calculate the position of the obstacle relative to one’s own position in space. Humans and many other animals can do this, but it wasn’t thought that simpler organisms such as zebrafish could do it.

Instead, many neuroscientists believed that visual perception of zebrafish was similar to that of organisms such as the simple worm C. elegans. In those worms, light detected by photosensitive cells can trigger reflexive responses such as moving toward or away from the light.

To explore the question of whether zebrafish can create mental representations of their 3D environment, Bolton created an experimental setup where the fish would need to try to avoid an obstacle blocking one of their possible escape paths. The experiments were done in the lab of Florian Engert, a Harvard professor of molecular and cellular biology, who is also an author of the study.

Each fish was placed in a circular dish about 12 centimeters in diameter, where they could swim freely. When a metal rod was dropped onto the dish, creating a loud bang, the fish would immediately flee. The researchers first showed that if no barriers were present, the fish would randomly choose either the left or the right as an escape path.

Then, the researchers placed a 12-millimeter plastic barrier blocking one of the escape routes. When a barrier was in place, the researchers found that the fish almost always chose to escape in the direction with no barrier, as long as there was enough light for them to see it. Furthermore, the fish were more likely to try to avoid the barriers when they were closer, suggesting that they are also able to calculate the distance to the barriers.

The zebrafishes’ quick reaction time — about 10 milliseconds — suggests that the animals must “pre-compute” a map of the barrier location before they hear the sound. Conduction of visual information from the retina to the brain takes about 60 milliseconds in zebrafish, ruling out the possibility that the fish check for obstacles after hearing the loud bang.

“They can’t do the mapping in real-time, because the escape is too fast relative to the tap,” Bolton says. “They need to pre-map the environment before, just in case a predator or something mimicking a predator shows up.”

Modeling the brain

This kind of pre-mapping behavior has been seen in rodents and other mammals, but not in simpler vertebrates. The findings in zebrafish open up a new way to explore questions of how the brain creates models of the world, says Misha Ahrens, a senior group leader at the Howard Hughes Medical Institute Janelia Research Campus.

“This work shows beautifully how a small and deceptively simple-looking animal possesses remarkable behavioral and computational capabilities. They are not just input-output machines; instead, they possess a model of the world around them that is invisible to us until we carefully probe those internal models with a carefully designed trigger,” says Ahrens, who was not involved in the study.

Because the zebrafish brain is smaller and simpler than the mammalian brain, it can be more easily imaged and manipulated, down to the level of individual neurons. Previous researchers identified a single pair of neurons, known as Mauthner neurons, which appear to mediate the zebrafish response to the sound. This study’s neural circuit experiments found that visual input of the barrier excites the Mauthner neuron that induces escapes away from barrier.

The researchers now plan to explore what part of the zebrafish brain encodes representations of depth perception. Neuroscientists already have a good idea of how and where the mammalian brain maps two-dimensional places (in the superior colliculus, which is analogous to a zebrafish brain region called the optical tectum), but how the third dimension of depth is added is not well-understood.

“If, for example, we find the 3D representation in the larval zebrafish optical tectum, that would be a guide to where it might be in the superior colliculus or the visual pathways of mammals, including humans,” says Mansinghka, who leads the Probabilistic Computing Project in the Department of Brain and Cognitive Sciences and also has a dual appointment in MIT’s Computer Science and Artificial Intelligence Laboratory.

Mansinghka also hopes that the new findings will help convince some cognitive and systems neuroscientists, who view zebrafish as too simple to be useful for their purposes, to consider it as a model with the potential to integrate many different approaches that scientists now use to study the brain.

“Historically, there has been a lot of divergence between people who study cells, people who study brain circuits, people who do imaging, people who study behavior, people who study cognition, and people who study computation,” he says. “It’s hard to do integrative research that addresses all those levels simultaneously, but here we may have shown that there is an organism that could be used to study perceptual computations at many different levels and connect it to the underlying neurons.”

The research was funded by the National Institutes of Health.

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The scientific wonders of zebrafish for biomedical research

Translucent living laboratories let scientists see life processes unfold before their eyes.

Contributing Writer Ohio State Wexner Medical Center

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The next time you visit an aquarium, look closely at the zebrafish. Those striped swimmers, just an inch or two long, might not look like it, but they carry the potential to revolutionize cancer treatment, enable humans to grow new heart tissue and contribute to countless other medical and scientific breakthroughs.

Zebrafish, or Danio rerio, live naturally in warm, shallow streams and rice paddies where India, Bangladesh and Nepal come together. To the fish hobbyist, they make an attractive and low-maintenance addition to the home tank.

But at The Ohio State University Wexner Medical Center, zebrafish are helping scientists push the boundaries of molecular genetics — revealing secrets and answering questions that could lead to innovations  and therapies yet unimagined.

From its origins at the University of Oregon more than 40 years ago, the zebrafish research model has spread worldwide, and Ohio State, with six independent zebrafish labs across campus, is a major player.

“We have several groups using the model to study related aspects of development and disease,” says Sharon Amacher, PhD , a professor with appointments in the College of Arts and Sciences’ Department of Molecular Genetics and the College of Medicine’s Department of Biological Chemistry and Pharmacology. “We really synergize. We meet regularly to share our research.”

Each of Ohio State’s zebrafish labs has a roster of between four and 12 people, for a total of about 50 researchers actively engaged in the work.

Three nurseries, six labs and endless genetic possibilities

Though zebrafish don’t look much like humans, it turns out we’re put together the same in most of the ways that count, with brains, spinal circuits, major organs and about 70% of our DNA in common. Even better, if you consider only the human genes known to be implicated in disease, 84% of those have zebrafish equivalents. And zebrafish have further benefits as a research model: They breed quickly and easily, and they’re small enough to house in large numbers but large enough to allow medical procedures like transplantation. Another distinctive feature? The embryos are transparent, allowing researchers with powerful microscopes to see cell processes unfold in real time. Even zebrafish adults can be made transparent when they carry mutations in two key pigmentation genes.

“In the early days, the model attracted a lot of biologists who wanted to understand how an embryo is put together and how embryos develop, how cells know what to do and when to do it,” Dr. Amacher says.

“But now, zebrafish are great for other studies, too. They get the same diseases humans do, so they’re very important in cancer studies,” Dr. Amacher says.

They’re also amazing at regeneration — a superpower that could have stunning implications for humans. For instance, when a human has a heart attack, those heart cells die and are never replaced. With zebrafish, 50% of the muscle cells of their heart can be destroyed and they will regenerate them within a month or two.

Zebrafish regeneration doesn’t stop with heart muscles. When a zebrafish spinal cord is severed, researchers say, that same fish will be swimming around in about six weeks, indistinguishable from their tankmates.

Those tankmates coexist by the thousands in three separate facilities on the medical center campus. Each is a sort of living genetic library, with floor-to-ceiling racks of 3- to 9-liter plastic tanks that are temperature controlled, automatically aerated, and recirculated several times per hour. The fish are grouped by age and genetic makeup, for cultivation of whatever traits are desired by the researchers.

In one campus facility, Dr. Amacher’s lab studies skeletal muscle and vertebral patterning, using the zebrafish as a model for vertebrates (animals that, like us, have spinal columns).

In another fish facility, the transparency of zebrafish embryos allows Martin Haesemeyer, PhD , assistant professor in the Department of Neuroscience at the College of Medicine, to actually see cells at work. Rather than employing electrodes to record electrical activity in neurons, he uses fluorescent proteins that report a change in a cell’s calcium level. This allows Dr. Haesemeyer to directly observe the cell growing brighter or dimmer with each change in activity.

Research possibilities

 Zebrafish live in warm, shallow streams in Asia. For medical researchers, this fish holds marvelous abilities.

Zebrafish and humans have a lot in common, including organs, spines and roughly 70% of the same DNA.

Translucent skin

Zebrafish embryos are translucent, allowing scientists to watch medical processes unfold before their eyes.

Ability to regenerate

Zebrafish have an astonishing ability to regenerate parts of their body, including heart muscle and even spinal cord tissue.

Dr. Haesemeyer is interested in thermoregulation, or the means by which animals maintain an optimal body temperature. Humans rely on involuntary processes such as sweating or constriction of blood vessels, but we also can use behavior — fan ourselves, say, or put on a sweater.

Fish, including zebrafish, lack any internal means of thermoregulation, making them ideal for the study of thermoregulation via behavior. In Dr. Haesemeyer’s words, “How does the animal find its comfortable temperature? How does it know which direction to swim?”

Basic research key to scientific progress

Better understanding that function, Dr. Haesemeyer explains, will help us understand how brains control things. Figuring out how thermoregulation works is important, he says, because when it goes awry, “There are very adverse consequences.”

“Knowing how it works in a healthy situation could help with fixing it when it’s broken,” he says.

It’s a common theme in the basic research done with the zebrafish model: Deciphering and describing a healthy system provides clues to managing an unhealthy one. “Diagnosing what’s wrong is so much easier if you know what ‘right’ is supposed to look like. It’s my pet peeve [that] so often in science we go after the disease first,” Dr. Haesemeyer says.

James Jontes, PhD , another zebrafish principal investigator who shares a facility with Dr. Haesemeyer, agrees.

Dr. Jontes, an associate professor in the Department of Neuroscience at the College of Medicine, studies cell adhesion molecules, a family of molecules that cause cells to stick together. He uses timelapse imaging to study the formation of neural networks in the brain.

“We can watch everything happen from the very first cell division, 10 minutes after [zebrafish embryos] are fertilized,” he says. “In two days, they’re swimming in short bursts; by five days, they’re hunting little microorganisms.”

Aaron Goldman, PhD , an assistant professor in the College of Medicine Department of Biological Chemistry and Pharmacology, focuses his research on the zebrafish’s “profound ability to regenerate damaged heart muscle.” His goal is to learn more about what distinguishes this remarkable ability in zebrafish from the scarring and muscle dysfunction that occur in humans.

Ohio State’s newest zebrafish researcher, Lihua Ye, PhD , is in the second year of a five-year career development award from the National Institute of Diabetes and Digestive and Kidney Diseases. She’s an assistant professor in the Department of Neuroscience at the College of Medicine, and for her, zebrafish offer an ideal window on the gut-brain axis. That’s a two-way system of communication between the brain and the part of the nervous system that regulates immune and endocrine functions. This web of neurons is embedded in the wall of the gastrointestinal system from the esophagus to the rectum.

With a zebrafish embryo under a microscope, Dr. Ye can introduce bacteria to the gut and see, in real time, the brain’s response. She sees potential applications for her work such as discovering how the human body interprets and responds to what a person consumes and how gut bacteria regulate our food choices.

Dr. Ye’s work benefits from yet another zebrafish attribute: their ease of breeding.

“The fact that we can very easily generate germ-free fish and manipulate the composition of the zebrafish gut microbiome is very powerful,” she says.

She shares a fish facility with Bradley Blaser MD, PhD , a member of the Leukemia Research Program at The Ohio State University Comprehensive Cancer Center — Arthur G. James Cancer Hospital and Richard J. Solove Research Institute and assistant professor of hematology at the College of Medicine. Dr. Blaser’s lab examines how blood stem cells respond in development under normal conditions and under induced stress. The work could explain how changes in the genetic makeup of a person’s blood over a lifetime may contribute to certain blood cancers.

A seventh Ohio State-affiliated researcher using the zebrafish model is Genevieve Kendall, PhD , who has her lab at Nationwide Children’s Hospital in the Center for Childhood Cancer and also is an assistant professor of pediatrics in the College of Medicine. Her work focuses on the relationship between certain childhood cancers and a misregulation in the development of skeletal muscle.

Science moves forward on the work of many minds

Laboratory researchers like Ohio State’s zebrafish scientists expect that their findings will eventually be applied to solving clinical problems.

“Translational value is always the goal,” Dr. Ye says. But they know that world-changing breakthroughs typically are built on many years of the basic research that they and hundreds of other scientists are doing at the College of Medicine.

Dr. Jontes understands that people affected by a disease urgently desire a cure. But he argues we wouldn’t learn as much if we were just simply focused through the lens of a specific disease. An institution such as Ohio State, with dozens of labs asking a diverse array of questions, is primed for discovery because the value in science is having “a lot of people out there doing a lot of different things.”

“Ninety-nine percent of the time, that’s how things work,” he says. “Collectively, we make progress.”

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Could a tiny fish hold the key to curing blindness?

Image courtesy of Beth Harvey, University of Pennsylvania

Imagine this: A patient learns that they are losing their sight because an eye disease has damaged crucial cells in their retina. Then, under the care of their doctor, they simply grow some new retinal cells, restoring their vision.

Although science hasn’t yet delivered this happy ending, researchers are working on it – with help from the humble zebrafish. When a zebrafish loses its retinal cells, it grows new ones. This observation has encouraged scientists to try hacking the zebrafish’s innate regenerative capacity to learn how to treat human disease. That is why among the National Eye Institute’s 1,200 active research projects, nearly 80 incorporate zebrafish.

How the Zebrafish Could Help Us Treat Eye Disease or Injury

The retina is a layer of tissue in the back of the eye that responds to light. But many scientists think of the retina as part of the brain. Like other neurons of the central nervous system, retinal neurons typically don’t replicate in adult humans. Loss of retinal neurons typically results in irreversible vision loss.  

Image credit: National Eye Institute

However, zebrafish, like newts, frogs, and a strange fish-like salamander called the axolotl, can regrow a variety of body parts – not only retinal neurons, but also the heart, fins, pancreas, brain, spinal cord, and kidney.

Zebrafish have a variety of traits that make them a great model for studying tissue regeneration: They’re capable of reproducting hundreds of offspring at a time. They’re cheap to maintain and express about 70% of the same genes that humans do. Unlike mice, which develop in a womb, zebrafish develop externally where scientists can easily observe them. And their flesh is nearly transparent during development, enabling researchers to observe their internal organs.

Scientists have long known that when zebrafish retinas are damaged, neuronal support cells called Müller glia start dividing to create neuronal precursor cells, which go on to become replacement retinal neurons. More recently, scientists have been trying to unravel the biological factors that initiate this process. Progress in that effort is detailed in several NEI-supported research projects over the past three years.

Studying zebrafish, James Patton of Vanderbilt University and colleagues found that when levels of the neurotransmitter GABA decrease, neural stem cells activate , these cells then migrate to  damaged retina and develop (differentiate) into whatever cell type is needed for repair. Patton’s findings help identify cues that stimulate zebrafish regeneration.

Jeff Mumm, Johns Hopkins University, reported that immune cells in the retina called microglia are necessary for zebrafish Müller glia to initiate regeneration after injury . After selectively knocking out microglia with a toxic enzyme, Mumm found that the Müller glia showed almost no regenerative activity after three days of recovery, compared with approximately 75 percent regeneration in control zebrafish. However, when an immunosuppressant was applied to inhibit microglia reactivity a day after retinal cell loss had begun, the pace of retinal neuron replacement accelerated. This observation suggests that microglia play different roles at different stages of injury/regeneration.

Jeff Mumm, Johns Hopkins, and collaborators, fluorescently labeled immune cells in zebrafish larvae to track immune system activity in a model of retinal degeneration. Image credit: Credit: David White, Mumm Lab, Johns Hopkins University School of Medicine

Findings in zebrafish by these other groups led Tom Reh at the University of Washington to unlock the regenerative potential of cells in the mouse retina . In newborn mice, the gene regulatory factor Ascl1 can direct Muller glia to become retinal neurons. This gene goes dormant when mice mature. By artificially expressing the Ascl1 gene in adult mouse Muller glia, Reh’s team turned the gene program back on, showing for the first time that Müller glia in the adult mouse can give rise to new functional neurons after injury. These neurons have the gene expression pattern, the morphology, the electrophysiology, and the epigenetic program to look like interneurons instead of glia, according to the report, and connect with the existing retinal circuitry and respond to light.

A second major challenge of regenerating the visual system is figuring out how replacement neurons in zebrafish find their way back to visual centers of the brain. The light-sensing photoreceptors connect to retinal ganglion cells (RGCs). RGC cell fibers called axons coalesce within the optic nerve where they exit the eye and disperse throughout the brain.

Beth Harvey, a postdoctoral researcher working with Michael Granato at the University of Pennsylvania, has developed a model for studying this process. 1 She uses zebrafish at the late larval stage so that she can observe RGC axons navigate to their appropriate brain target after injury, using a technique called confocal microscopy. Interestingly, she found that axons are more likely to innervate appropriate targets when the optic nerve is only partially cut—like leaving a trail of breadcrumbs for regenerating axons to follow.

Illustration of zebrafish head showing optic nerve, eye, and brain. Image courtesy of Beth Harvey, University of Pennsylvania.

To accelerate progress, the NEI funded a consortium of scientists as part of its Audacious Goals Initiative to identify biological factors that affect the restoration of functional connections within the retina and between the eye and brain. Projects within the consortium have used various models to evaluate hundreds of genes for their role in regeneration as well as compounds that modify their activity. In partnership with Michael Dyer from St. Jude’s Children’s Hospital, the NEI is uploading consortium data to an online database to help future investigations.

1. Harvey, B. M., Baxter, M. & Granato, M. Optic nerve regeneration in larval zebrafish exhibits spontaneous capacity for retinotopic but not tectum specific axon targeting. PLoS One 14, e0218667, doi:10.1371/journal.pone.0218667 (2019). Pubmed

This press release describes a basic research finding. Basic research increases our understanding of human behavior and biology, which is foundational to advancing new and better ways to prevent, diagnose, and treat disease. Science is an unpredictable and incremental process— each research advance builds on past discoveries, often in unexpected ways. Most clinical advances would not be possible without the knowledge of fundamental basic research.

NEI leads the federal government’s research on the visual system and eye diseases. NEI supports basic and clinical science programs to develop sight-saving treatments and address special needs of people with vision loss. For more information, visit https://www.nei.nih.gov .

About the National Institutes of Health (NIH): NIH, the nation’s medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit https://www.nih.gov/ .

NIH…Turning Discovery Into Health®

• Open access
• Published: 07 May 2020

Zebrafish as an alternative animal model in human and animal vaccination research

• Ricardo Lacava Bailone 1 , 2 ,
• Hirla Costa Silva Fukushima 3 ,
• Bianca Helena Ventura Fernandes 4 ,
• Luís Kluwe De Aguiar 5 ,
• Tatiana Corrêa 6 ,
• Helena Janke 6 ,
• Princia Grejo Setti 6 ,
• Roberto De Oliveira Roça 2 &
• Ricardo Carneiro Borra 6  

Laboratory Animal Research volume  36 , Article number:  13 ( 2020 ) Cite this article

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Much of medical research relies on animal models to deepen knowledge of the causes of animal and human diseases, as well as to enable the development of innovative therapies. Despite rodents being the most widely used research model worldwide, in recent decades, the use of the zebrafish ( Danio rerio ) model has exponentially been adopted among the scientific community. This is because such a small tropical freshwater teleost fish has crucial genetic, anatomical and physiological homology with mammals. Therefore, zebrafish constitutes an excellent experimental model for behavioral, genetic and toxicological studies which unravels the mechanism of various human diseases. Furthermore, it serves well to test new therapeutic agents, such as the safety of new vaccines. The aim of this review was to provide a systematic literature review on the most recent studies carried out on the topic. It presents numerous advantages of this type of animal model in tests of efficacy and safety of both animal and human vaccines, thus highlighting gains in time and cost reduction of research and analyzes.

Introduction

The role of the immune system is to protect a body against bacterial, viral, or any foreign antigen invasions. In order to improve protection, vaccination is used to boost immunity against diseases caused by microorganisms. It typically contains a less virulent agent that triggers a reaction, thus, stimulating a body’s immune system to recognize it as foreign. In the process, a body’s defense mechanism learns to recognize and destroy a microorganism, its toxins or surface proteins [ 94 ] every time an invasion is identified. The use of vaccination is important because it promotes the stimulation of the body’s defense mechanisms and the development of both individual and collective immunity. Vaccination can act on specific (adaptive) and nonspecific (innate) immune responses unlike immunostimulants which only act on innate response. In addition, it should be noted the role vaccines play in controlling diseases as preventative as well as non-therapeutic measures. Therefore, the body is able to produce antibodies that recognize, signal and neutralize pathogens or particular cellular responses which detect the specific antigens with high efficiency and affinity. As a result, vaccines protect the body against future infections [ 27 ] thus reducing the need for the use of antibiotics and other types of drugs.

Despite the study of immunology in fish being more recent compared to those of humans and in animals, the concepts and techniques used are similar [ 60 ]. The study of the use of vaccines in fish is an area of fast-growing. As aquaculture expands and the need to control pathogens becomes more pressing, the commercial vaccination of different varieties of fish is already a reality in many countries. It aids in the prevention of diseases that could pose health risks to the shoal as well as in avoiding the economic losses due to mortality caused by infection. It reduces the contamination of water bodies by the excessive use of antibiotics, and the reduction of final fish product quality [ 5 , 24 , 42 , 79 , 100 ].

The Zebrafish model has been widely used in both animal and human health research and, more recently, in aquaculture too. In spite of rodents being the most widely used research model in the world, in recent decades the use of the zebrafish ( Danio rerio ) model has exponentially increased among the scientific community. It follows the principle of 3Rs (replacement, reduction, and refinement) as required by a multiplicity of national and international regulatory bodies. Furthermore, the use of zebrafish model results in a reduction of time and use of resources when compared to those more established animals’ models. It also provides a greater informational and predictive capacity when compared to in vitro results [ 53 ]. Thus, using the zebrafish model, it is possible to replace and reduce the use of mammals in research as well as mitigate problems related to the welfare of those animals. Furthermore, zebrafish is used as confirmatory models of the positive previously obtained results, thus, having the ability to refine the findings [ 2 ]. A review of the literature was carried out aiming at presenting the most recent information on vaccination of fish, which brings to light the advantages of this animal model in tests of efficacy and safety of both animal and human vaccines.

Material and methods

The present study was based on a systematic literature review carried out using databases such as Science Direct, Google Scholar and SciELO (Scientific Electronic Library Online). Emphasis was given on identifying publications using search words and terms containing ‘human vaccination’ and ‘animal vaccination’. Particularly, the main key-words searched included ‘Zebrafish model’, ‘vaccine safety’, ‘diseases’, ‘infection’ and ‘toxicology’. Initially, 99 publications were identified which included books, rulings and articles published by international scientific journals of high impact factor. The publications were selected according to relevance and timeliness. 19% of the articles used were published in the last year, 65% in the last 5 years, and 89% published in the last 10 years.

Zebrafish model and vaccines testing

Vaccination safety.

When devising immunization experiments, challenge trials for vaccine development evaluate the efficacy and safety of the vaccine against different pathogens. These are normally assessed using animal models, mainly mammals, which are often imprecise in reflecting human diseases [ 93 ], not to mention time consuming, and require a large number of animals. Moreover, the mortality and clinical signs as well as laboratory tests are usually analyzed to evaluate the innate (non-specific) or adaptive (specific) immune system response. As in mammals, Zebrafish has a well-maintained adaptive immune system composed of T and B lymphocytes that develop from the thymus and kidneys respectively. However, in relation to the development of memory lymphocytes, fish seem to have memory cells of the type B and T [ 78 ]. Yet, there has not been enough data to confirm that in Zebrafish. Zebrafish also presents the enzyme system involved in the process of genetic rearrangement that originates the B (BCR) and T (TCR) lymphocyte receptors. As in humans, Zebrafish has recombination activator genes that control the rearrangement of gene segments V, D and J to produce the diversity of antibodies and lymphocyte receptors. In addition, the zebrafish’s immune system has only approximately 300,000 antibody-producing B cells, making it three orders of magnitude smaller than mice and five orders simpler than humans [ 48 ].

The efficiency of the humoral response increases due to the increased affinity of the antibodies. Affinity maturation of antibody responses is less efficient in cold-blooded vertebrates compared to mammals. Despite this, in zebrafish, data revealed that specific nucleotides in regions of the BCR receptor were target of directed mutations. Therefore it was suggested that activation-induced deaminase and affinity maturation contributed to the diversification of antibodies also in fish [ 56 ]. Immunization of teleost fish with the TNP-KLH antigen (linked to trinitrophenyl to keyhole limpet hemocyanin), for example, induced the production of specific low affinity antibodies, which were replaced in 5 weeks by antibodies of intermediate affinity, and after 15 weeks, by antibodies with greater affinity for the antigen [ 28 , 97 ].

Among the immunological tests, the most frequent ones are: complete hematological analysis by counting erythrocytes; thrombocytes and leukocytes; differential white cell count; hematocrit; glucose; organ histology, and immunological essays such as serology, specific antibody titration, and agglutination [ 4 , 29 , 57 ]. Furthermore, toxicity tests can be also conducted using zebrafish such as embryotoxicity, hepatotoxicity, neurotoxicity, endocrine toxicity, genotoxicity, among others as proposed by Bailone et al. [ 3 ].

Up to now, these tests have been conducted using rodents, but in recent decades, the Zebrafish model has proved to be an important tool in the studies of infections and immunological responses. This model has the advantage of having OECD-specific guidelines for safety evaluation of chemical compounds (acute toxicity), which is performed within 96 h [ 65 ]. In addition, observations can be made in real-time allowing for the monitoring of embryogenesis (Fig.  1 ) as well as regarding the effects of vaccines in relation to cardiovascular, hepatic, nervous, and endocrine, not to mention, behavioral aspects too [ 18 , 40 ].

Embryos of zebrafish 0, 6, 24 and 48 h’ post-fertilization. Larvae of zebrafish 72 and 96 h post-fertilization

Prior to vaccines being tested on humans, livestock or pets, these should be assessed using animal models to avoid causing them harm, including death, especially in the case of immunosuppressed organisms, children and the elderly [ 26 ]. As for vaccination in humans, for example, about 0.4 to 1.9 people per million who had been vaccinated with BCG against tuberculosis may have developed the disease through vaccine contagion. For hepatitis B, 1 in 600,000 people vaccinated may have presented a severe allergic reaction (anaphylaxis). In the case of vaccine against poliomyelitis, vaccine contagion happened to 1 in every 3.6 million vaccinated. Moreover, to combat yellow fever, the vaccine contagion and seizures happened to 1 in 22 million and internal hemorrhages happened to 1 in 450,000. Thence, the occurrence of side effects is very rare. Side effect reactions in humans may also be observed to be caused by other vaccines such as yellow fever, measles, mumps, rubella, chicken pox, diphtheria and tetanus. The most common symptoms are seizures, severe allergic reactions, meningitis, encephalitis [ 26 ]. Although these risks are irrelevant when compared to damages that could be caused by the non-use of a vaccine, the toxicology, the side effects and immunization at different concentrations ought to be adequately tested.

Thus, the Zebrafish model has the advantage of a researcher to follow in real-time the fish’s development from its embryogenesis to full organ development which is reached about 36 h after fertilization. This allows for a vaccine’s effect on all the major organs precursors to be closely studied [ 53 ] such as using immunohistology (Fig.  2 ).

Histology of adult zebrafish (hematoxylin eosin). a Male. b Female

Zebrafish and mammalian toxicity (Lethal concentration – LC 50 ) profiles are surprisingly similar for a range of substances specified in Table  1 below. Therefore, toxicity studies support the effectiveness of using the zebrafish model for the purpose of testing these substances. Furthermore, they can be extrapolated to the active ingredients present in the vaccine, and enabling quick parallel studies of vaccine reactions in humans and zebrafish.

Advantages of zebrafish model in vaccination tests

Compared to other vertebrates, zebrafish have extra biological advantages including high fecundity, external fertilization, optical transparency and rapid development. Moreover, Zebrafish possess a highly developed immune system that is remarkably similar to the human one. Therefore, it is expected that the majority of the signaling pathways and molecules involved in the immune response of mammals would also exist and behave similarly in fish [ 89 ]. Consequently, the presence in fish of elements of innate and adaptive immunity enables research in infectious processes, being susceptible to infections by gram-negative and gram-positive bacteria, protozoa, viruses, fungi and mycobacteria.

The development of special cloning, mutagenesis and transgenesis techniques allowed the identification of a significant number of mutants. Commercial mutant zebrafish lines and the recently developed CRISPR/Cas9 genome modification system provide the means to create knockout zebrafish for studying individual genes at a whole organism level [ 66 ]. Non-pigmenting mutants such as Casper zebrafish have also helped improve visibility of internal organs [ 92 ]. In addition it is easy to generate transgenic zebrafish with ‘reporter genes’ to facilitate analysis in live fish [ 87 ]. Because the zebrafish genome is conserved in humans, information obtained from zebrafish studies may lead to translational results in humans [ 38 ].

Examples of mutant animals displaying human-like diseases are numerous such as: sapje, which has the gene homologous to that of Duchenne muscular dystrophy; dracula , related to erythropoietic protoporphyria; van Gogh, model of the DiGeorge syndrome; and gridlock , which induces coarctation of the aorta [ 47 ]. Research in tumor suppressor genes p53 and apc ( adenomatous polyposis coli) is another area of great interest . The importance of the p53 gene in human carcinogenesis is well recognized and recent studies have shown zebrafish as an excellent model for assessing the presence (or not) of gene stability. Lymphoid leukemia, melanoma and hepato-carcinoma have already been described in zebrafish thus confirming that the molecular mechanisms involved are similar to those of humans [ 49 ].

Regarding the administration of vaccines, in view of the different routes of applications presented in animals and humans, the zebrafish model still allows the immunization of embryos, facilitated by its transparency, using glass needles (Figs.  3 and 4 ). Interestingly, the fact that the fish’s adaptive immune system does not reach maturity up to 4 weeks after fertilization allows them to be used without the need for immunosuppression in the embryonic stages [ 32 ] in the case, for example, of tumor xenograft experiments.

a Vitelline Yolk Injection (24 HPF), Magnifying Glass Nikon SMZ745, 50X; B) Vitelline Yolk Injection (24 h.p.f.), Magnifying Glass Nikon SMZ745, 50X

a 24 HPF Zebrafish Embryo Brain Injection, Nikon Microscope; b Brain injection of turbo-red substance into a 24 HPF zebrafish embryo; c Luciferin-labeled 4 T1 tumor cell bioluminescence in 3-month-old animals

In zebrafish larvae, a rapid systemic infection can be initiated by direct microinjection of a bacterial suspension into the bloodstream. Alternatively, a more localized infection may be induced by the injection of microbes into the muscle tail or the hindbrain ventricle [ 6 ]. For high transfer rate, the microbes can be readily injected into the yolk for the first few hours after fertilization. However, it is important to keep in mind that the yolk lacks immune cells, and therefore the bacteria are able to grow freely before invading the larval tissues [ 51 ].

Several transgenic zebrafish lines containing fluorescent markers in different cells of the immune system have been developed to visualize host-microbe interactions in the transparent larvae. For example, recruitment of fluorescent neutrophils to the site of bacterial infection (which can also be labeled with fluorescence) could be easily followed and quantified in real time. Yet, so far, researchers have focused primarily on larval infection patterns [ 51 ].

Fish vaccines

In the prevention of disease outbreaks causing mortalities in aquaculture, similarly to any other animal production system, vaccination is essential. Thus, the use of vaccines for that purpose could be improved based on the results from the studies performed in zebrafish [ 89 ]. The development of vaccines for aquaculture has been an important milestone for guaranteeing a continuous safe and high standard animal health production system. In recent years, zebrafish models have been chosen as the preferred model in the production of fish vaccination experiments against several pathogens that cause losses in aquaculture around the world such as bacteriosis and viruses. One of the most important pathogen studies applied to fishing production is attributed to Guo et al. [ 35 ]. They analyzed the protective efficacy of four iron-related recombinant proteins and their single-walled carbon nanotube encapsulated counterparts against the Aeromonas hydrophila infection in zebrafish. They observed that the immune response was increased after vaccination. Guo et al. [ 34 ] also studied Edwardsiella tarda which is an important intracellular pathogenic bacterium that causes the infectious disease Edwardsiellosis in fish. They proved that live E. tarda vaccine enhanced innate immunity by metabolic modulation in zebrafish.

Vibrio anguillarum , a bacterium that causes vibriosis, was also studied by Ye et al. [ 98 ] who observed the maternal transfer and protection role in zebrafish offspring following vaccination of the brood stock with a live attenuated V. anguillarum vaccine. They proved that the development of immune cells was enhanced and the maternally-derived antibody could protect early embryos and larvae from the attack of specific pathogens via vaccination with a live attenuated vaccine. Furthermore, Liu et al. [ 50 ] analyzed the profiling immune response in zebrafish intestine, skin, spleen and kidney when immersion vaccinated was used with a live attenuated V. anguillarum vaccine. Immersion, or bath vaccination, is a common practice in aquaculture, because of it being convenient as mass vaccination giving sufficient protection. The fish is submerged in water with a sub lethal concentration of the bacteria for a specific time. Liu et al. [ 50 ] observed that antibodies were either produced at antigen-contact tissues or in immune organs. Zhang et al. [ 101 ] studied Th17-like immune response in fish mucosal tissues after administration of live attenuated V. anguillarum via different vaccination routes. When compared to injection vaccination, immersion vaccination elicited intense Th17-like immune responses in the gut tissue of zebrafish. Vibrio vulnificus , that is an aquatic pathogen that can cause primary sepsis and soft tissue infection, was also tested during an experimentation of zebrafish’s reaction to vaccine. It was concluded that CpG oligodeoxynucleotides, a type of essential immunomodulators, protected zebrafish against Vibrio vulnificus induced infection [ 15 ].

Francisella noatunensis is a bacterium that causes granulomatous disease in freshwater and marine fish, and remains an unsolved problem for the aquaculture sector as no efficient vaccines are yet available. Lagos et al. [ 46 ] studied the immunomodulatory properties of Concholepas concholepas hemocyanin against francisellosis in a zebrafish model, proving that his adjuvant was a potential one for aquaculture vaccines. Moreover, Brudal et al. [ 11 ] observed that vaccination with outer membrane vesicles from F. noatunensis reduced the development of francisellosis in a zebrafish model.

Streptococcus sp. has also been studied with the Zebrafish model. Streptococcus parauberis is the major infectious agent of streptococcosis in olive flounder ( Paralichthys olivaceus ). Kim et al. [ 45 ], studying the identification of novel immunogenic proteins against S. parauberis by reverse vaccinology using zebrafish model, identified 41 vaccine candidates against S. parauberis. Furthermore, Streptococcus iniae was studied by Membrebe et al. [ 58 ] testing the protective efficacy of Streptococcus iniae derived enolase against Streptococcal infection in zebrafish model. In that study, enolase protein was evaluated to induce cross-protective immunity against S. iniae and S. parauberis which are major pathogens causing streptococcosis in fish.

Further to the aforementioned examples, many other diseases have been investigated with the Zebrafish model. For example, Rhabdovirus, which is one of the most important diseases in salmonids, is a virus that causes hemorrhagic viral septicemia [ 44 , 64 ]. Listeria monocytogenes [ 19 , 20 ]; Piscirickettsia salmonis which causes salmonid rickettsia sepsis (Tandberg et al. [ 83 ]); and in adjuvant test to improve the efficacy of vaccines [ 44 ], among others [ 82 ].

Animals and human vaccines

The zebrafish model has been used not only in aquaculture, but also in veterinary and human medicine. So far, it has become one of the major model systems used in modern biomedical research [ 51 ]. According to Torraca et al. [ 86 ], zebrafish can be also used as a model for pathogenesis and host defense, modeling many human diseases, such as tuberculosis, Staphylococcus aureus and Shigella infection, among others, as well as model to investigate immune cells, infection and inflammation of different kind of human diseases.

Torraca et al. [ 86 ] posited that zebrafish could also be used as a model for Tuberculosis which is a devastating infectious disease worldwide and with no current prospect of efficient prevention. Tuberculosis is an infectious disease caused by bacilli from the Mycobacterium tuberculosis complex. It is estimated that up to one third of the world’s population is infected with M. tuberculosis and have active tuberculosis, which often develops decades after the primary infection. Annually about two million people perish of tuberculosis and, so far, due to the lack of well-established animal models, such a disease has been difficult to study [ 51 ].

An infection by Mycobacterium marinum in adult zebrafish resembles that of human tuberculosis, as demonstrated by Myllymäki et al. [ 62 ]. Those authors proved that the M. marinum infection model in adult zebrafish was suitable for preclinical screening of tuberculosis immune’s responses and vaccines. It was also a promising new model for tuberculosis vaccine research, including the pre-clinical identification of vaccine antigens [ 16 , 17 , 36 , 41 , 61 , 67 ];). Other species of Mycobacterium have also been studied, such as M. bovis [ 52 , 73 ] and M. abscessos [ 7 ]. M. bovis is most common in cattle, but also affects humans. M. bovis Bacillus Calmette-Guérin vaccine is currently available as a prophylactic tool for preventing the disease. It has been shown to be efficient in preventing disseminated forms of tuberculosis in children; however, its efficiency is limited in areas where individuals have had prior exposure to environmental mycobacteria, and its efficacy decreased with a host’s age [ 55 ].

Moreover, teleost models offer an expanding platform for the understanding of mycobacterial infections and those mechanisms that offer the greatest potential to enhance host protection [ 37 ]. The models make it possible to screen the host and bacterial factors that modify the disease and facilitate the search for new therapeutic agents. It has recently been shown that zebrafish can also be used for the potential screening of DNA-based vaccines and, in particular, for identifying novel antigens protecting against mycobacteria [ 67 ]. Therefore, using the Zebrafish model is expected to accelerate the understanding of the pathogenesis of tuberculosis which would lead to the development of better vaccines. Yet, the usefulness of this model is not limited to tuberculosis, which as seen before it could benefit research for many other important infectious diseases [ 51 ].

Similarly, this model also helps to elucidate bacterial infections in animals and humans by Aeromonas hydrophila [ 91 ], Pseudomonas aeruginosa [ 74 ], Escherichia coli nonpathogenic [ 63 ], E. coli CFT073 [ 95 ], Listeria monocytogenes [ 80 , 81 ], Myroides odoratimimus [ 72 ], Cronobacter turicensis [ 25 ], Streptococcus agalactiae [ 70 , 96 ],  Streptococcus iniae and Streptococcus pyogenes [ 59 , 76 , 77 ], among others [ 12 , 85 ].

Shigella is a major cause of dysentery worldwide, accounting for up to 165 million cases of shigellosis each year [ 23 ]. Yet, despite there not existing vaccine available as yet, the human and animal challenge–rechallenge trials with virulent Shigella as well as observational studies in Shigella-endemic areas are promising. The incidence of the disease decreased following Shigella’s infection which pointsto a biological feasibility of a vaccine [ 54 ]. Phalipon et al. [ 71 ] as well as Mani et al. [ 54 ] proposed that adult zebrafish could be used to study the immune response to Shigella, which is crucial to understanding the crosstalk between Shigella and T-lymphocytes [ 75 ] thus this being relevant in the development of vaccine strategies. Studies have also been conducted with Zebrafish model to promote a vaccine against Salmonella, which produces gastroenteritis that causes massive morbidity and mortality in adults and children in developing countries. Howlader et al. [ 39 ] proved that zebrafish was an excellent model for the study of vaccines using successive immersion triple vaccines with the single serotype Salmonella. Typhimurium and Salmonella entereditis induced protective efficacy against a high dose (10 8  CFU/ml) of infection by these pathogens.

Other microorganisms of importance such as fungi which can cause pathologies in humans, such as Candida albicans [ 10 ], Cryptococcus neoformans [ 8 , 84 ] and Mucor circinelloides [ 90 ] have also been the subject of study with teleosts. In addition, viruses such as Herpes simplex [ 13 , 31 ]; human norovirus [ 88 ]; Vesicular stomatitis [ 33 ]; hepatite C [ 21 , 22 ]; Chikungunya [ 1 , 9 , 14 , 68 ]; Sindib [ 69 ] and Influenza A [ 30 ] are some of the human viruses already studied by the zebrafish model in both embryos and larvae.

Conclusions

The use of the Zebrafish model for the production of vaccines with application for both animals and humans, despite already being a reality, is still underused. This model is an important tool for the development of new safe vaccines against diseases which do not yet have preventive treatment, or for which the existing vaccines are not so effective. Thus, previous screening tests with zebrafish have been proven to be effective in preliminary phases prior to testing with mammalians. Despite the evidence from the literature indicating that science in this field is in its infancy, when compared to other animal models used in research, teleost models have proved to be effective in the elucidation of the infection and immunological responses to the diverse animal and human pathogens. In addition, the reduced financial cost and time frame needed for testing are another attractive regarding the use of zebrafish. Thus, it is expected its use would expand in the coming years.

Availability of data and materials

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Bailone, R.L., Fukushima, H.C.S., Ventura Fernandes, B. et al. Zebrafish as an alternative animal model in human and animal vaccination research. Lab Anim Res 36 , 13 (2020). https://doi.org/10.1186/s42826-020-00042-4

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The University of Maine’s zebrafish facility is a bastion of research 

In the two adjacent rooms, the air is thick with an almost tropical humidity and a quiet gurgling sound comes from a complex circulating water system. Inside, racks of tanks hold tens of thousands of tiny fish. They are all zebrafish — an inch-long relative of the minnow native to South Asia — but a close look reveals their variations: transparent “Casper” zebrafish; fish dyed with purple, red and green markers; and fish with spots instead of stripes, or long flowing fins in place of the stubbier standard set.

The impressive set-up comprises the University of Maine’s Zebrafish Facility, which was the first of its kind in Maine when it launched in 2003. Four years ago, it became a  Coordinated Operating Research Entities  (CORE) unit, providing research and development resources for Maine and beyond. 

UMaine was one of the first institutions in New England to use the zebrafish as a model organism for research looking at everything from muscular dystrophy to bacterial infections. Over the past two decades, researchers at the University of Maine have made groundbreaking discoveries with these tiny fish — and they are just getting started.

Zebrafish have many advantages as a model organism. They have been used for over a century to study developing embryos because their fertilization and development are external, and the embryos are translucent and easy to manipulate. Zebrafish genomes are similar to humans, particularly their immune systems, but they mature quickly, mate in large batches and their genes are easy to tinker with for genetic research. 

Compared to other common model organisms like mice, zebrafish are also inexpensive and space efficient. UMaine’s Zebrafish Facility is a testament to that, boasting 21 recirculating racks of tanks for genetics and development work in its main 20 by 30 foot room in Hitchner Hall, plus another room next door with seven racks of tanks and one isolated rack for disease and toxicology research. There are a total of 1,700 tanks 3- and 10-liter tanks throughout the system, with space for over 30,000 zebrafish.

Before the facility was set up in its current iteration, former UMaine professor Carol Kim had managed several tanks for her own research on the innate immune response to pathogens and environmental toxicants starting in 1999. Kim, who eventually served as the director of the Graduate School of Biomedical Science and Engineering and university’s Vice President for Research, spearheaded the effort to expand the facility to include more researchers.

Mark Nilan, the facility’s operations manager, worked with the UMaine Center for Cooperative Aquaculture Research to set up the lab in 2003 while completing his undergraduate degree in aquaculture (a program that was eventually restructured into a certificate in 2007). 

“In the early days when they set up these labs they would just put people who had a fish tank and that was a disaster,” Nilan says. “UMaine set up for success with its certification in aquaculture.”

Nilan said that current system is more analog than many modern zebrafish lab set ups — he balances the pH of the water himself instead of digitally, for example — but that hands-on approach is part of what he believes has kept the lab from having any major incidents of zebrafish mortality since he has managed it. He recruits students through the aquaculture minor at the School of Marine Sciences for daily “animal husbandry” tasks like cleaning tanks, performing maintenance and feeding the fish. The Institutional Animal Care and Use Committee regularly checks on the lab to ensure its animal subjects are treated ethically and humanely.

Though the facility in and of itself is impressive, the research and education done there is perhaps even more so. Robert Wheeler, associate professor in the Department of Molecular & Biomedical Sciences and faculty liaison for Zebrafish Facility, said that there are currently five labs at the University of Maine that are “100% committed to working with zebrafish.” His lab uses zebrafish to study fungal pathogens in hospital settings . Ben King uses zebrafish for genetics projects in his bioinformatics lab, while Melody Neely’s lab studies host-pathogen interactions during bacterial infections. Clarissa Henry uses zebrafish to study muscular dystrophy , and Jared Talbot’s lab uses zebrafish for research on muscle development (he has just won an award for his efforts to share zebrafish with the scientific community). 

The facility also supports teaching at the School of Biology and Ecology, Molecular and Biomedical Sciences at the Hutchinson Center in Belfast by, for example, providing zebrafish embryos to classes studying early development. Researchers across the country can order zebrafish embryos from the facility, too.

Since 2017, the UMaine zebrafish facility has earned over $7.2 million in extramural funding from the National Institutes of Health, National Science Foundation and Burroughs Wellcome Fund, as well as over $104,000 in intramural funding from the University of Maine and University of Maine System. Over 90 undergraduates have been trained through the lab for highly skilled jobs and graduate school at prestigious institutions, as well as 35 graduate students and 1 post-doctoral fellow. The facility has produced 42 peer-reviewed publications in journals like Nature, Cell Reports, Cell Host & Microbe and eLife. The facility has conducted community outreach with over 120 high school students. Students who collaborated with Henry to use the facility’s zebrafish won first place in eCYBERMISSION, a virtual STEM competition for grades 6-9.

Wheeler expects the success of the facility will only grow as zebrafish become an increasingly popular model organism. He says there are grants in the works to “significantly increase” the size of the UMaine Zebrafish Facility by adding more rooms for racks in Hitchner Hall.

“One of the things that happens when the community keeps growing is that there are more and more tools that get developed, more and more foundational knowledge that gets discovered. The combination fuels a more rapid science that attracts more people, builds more tools and this feeds back again.”

Contact: Sam Schipani, [email protected]

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The Zebrafish model in dermatology: an update for clinicians

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• Published: 17 June 2022
• Volume 13 , article number  48 , ( 2022 )

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• Irene Russo 1   na1 ,
• Emma Sartor 1   na1 ,
• Laura Fagotto 1   na1 ,
• Anna Colombo 1 ,
• Natascia Tiso 2   na2 &
• Mauro Alaibac 1   na2  

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Recently, the zebrafish has been established as one of the most important model organisms for medical research. Several studies have proved that there is a high level of similarity between human and zebrafish genomes, which encourages the use of zebrafish as a model for understanding human genetic disorders, including cancer. Interestingly, zebrafish skin shows several similarities to human skin, suggesting that this model organism is particularly suitable for the study of neoplastic and inflammatory skin disorders. This paper appraises the specific characteristics of zebrafish skin and describes the major applications of the zebrafish model in dermatological research.

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1 Introduction

Since their use, animal model systems have offered a technical means to perform studies that could not be otherwise undertaken in human subjects. They represent a fundamental part of research and offer precious keys to understanding human physiology and pathophysiology. Moreover, not only do they allow for more advanced pharmacokinetic and pharmacodynamic studies, but also for the discovery of new treatments for human diseases.

Because of their well-known physiological and genetic similarities to humans, murine and other mammalian models have been routinely used for medical research. However, other animal models are showing distinct advantages over these conventional models. Therefore, interest in this field has increased over recent years.

Among the animal models studied thus far, fish seem to be the most interesting non-mammalian vertebrate model, because of their low maintenance costs and ex uterus development of progeny that allows for in vivo imaging. For instance, Platyfish ( Xiphophorus ) and Medaka ( Oryzias latipes ) have already been successfully used as models to study melanoma [ 1 , 2 , 3 ].

2 The zebrafish organism

The zebrafish ( Danio rerio ) was first introduced as a model for genetic studies by Streisinger and colleagues in the early 1980s [ 4 ]. Zebrafish are small vertebrate tropical fish characterized by low-cost maintenance and exploited as a model for both reverse and forward genetic studies.

At first, zebrafish were used as a model in forward genetic studies (the identification of a specific genotype through observation of a certain phenotype). This was the case in N-ethyl-N-nitrosurea (ENU) induced mutagenesis used to produce point mutations, followed by extensive phenotypic screening. However, forward genetic screens using this method were time consuming and laborious [ 5 , 6 ].

Fortunately, increasingly advanced techniques in recent years have allowed to overcome the challenges of creating several disease models using zebrafish and to expand zebrafish studies through the reverse genetics (the observation of the phenotype produced by a known genotype). Moreover, studies have proved that there is a high level of similarity between human and zebrafish genomes, estimating that 70% of human genes have at least one zebrafish ortholog. These data are astonishing, especially if we consider that a mouse shares about 80% of its genome with humans [ 7 , 8 ]. Moreover, over 80% of known human disease genes, including oncogenes and tumor suppressor genes, have their orthologues in zebrafish and several pathways are also conserved, even those implicated in carcinogenesis [ 8 ].

All these characteristics and evidence have boosted the use of zebrafish as a model for understanding human genetic disorders, including cancer, and have pointed out their potential for in vivo screening for new therapies, which is especially important in our era of personalized medicine.

Therefore, although mouse models remain the most used in the medical research field, the zebrafish has several advantages and unique features that murine models do not have, which explain its ancillary and complementary role.

The advantages of using zebrafish as a model are numerous, going beyond their low-cost maintenance and small size. They display a high fecundity, with the ability to fertilize about 200–300 eggs every 5–7 days. Fast ex utero development together with the embryos’ optical clarity allow for the observation of early physiological and/or pathological development by in vivo direct cell imaging [ 9 , 10 ]. In addition, the casper zebrafish was introduced as a genetic strain intentionally created to maintain transparency throughout adult life, making it even more affordable to study cancer cells’ behavior in a living organism [ 11 ].

Cell-based assays for the study of the absorption, distribution, metabolism and toxicity of compounds and drugs give only limited information, whereas pharmacologic molecule screening in zebrafish might help to overcome this problem. Parallel physiological responses have been observed in the use of drugs and small molecules in zebrafish and mammal models [ 9 ]. Drug screenings benefit from both the embryos’ transparency, which makes it easy to collect imaging data after treatment, and the high throughput assays, which are made possible by the female’s ability to lay many eggs (about 10,000 eggs per annum). This means that imaging, cellular analysis and advanced statistics can be performed simultaneously in an incredibly large number of fish, with laboratory space being the only limiting factor [ 12 , 13 ]. These premises explain the zebrafish’s potential role as a bridge between cell-based assays and biological validation of a certain compound.

Moreover, zebrafish might represent a clue in the attempt to identify therapeutic targets for the treatment of human diseases, which remains a big challenge in medical science [ 14 ]. In phenotype-guided drug studies, the presence of phenotype alterations in the whole organism may suggest the effectiveness of a drug, even when the target is unknown. This approach helps both the development of new drugs and the simultaneous identification of the molecular pathways underlying the disease that are inducing that specific phenotype [ 15 ]. Compared to mice, zebrafish studies enable us to analyze a greater number of phenotypes at reduced costs and labor.

Even though this fish model is extremely versatile in medical and especially in pharmacological research, there are a few drawbacks that should also be pointed out. Firstly, the zebrafish is a poikilothermic fish that needs to be bred in an environment with a temperature around 28 °C to survive. This differs from mammals’ homeostatic temperature, thus hindering studies where temperature is a determining factor. However, it can tolerate a wide range of temperature variation, spanning between 6 and 38 °C, for limited periods of time [ 16 ]. Secondly, teleost genome duplication involves the presence of genes in more than one copy (paralogs), which might hamper molecular genetic studies. Lastly, another disadvantage of the zebrafish is the scarcity of available antibodies that specifically target zebrafish proteins, and the technical difficulty in raising antibodies against zebrafish targets [ 17 , 18 , 19 ]. This is especially relevant for cell surface and secreted proteins, since immunogenic glycans on zebrafish extracellular proteins hamper elicitation of protein-specific antibodies in mammals used for raising such antibodies [ 20 ].

2.1 Zebrafish skin

Fish skin comprises the epidermis, dermis, and hypodermis, thus resembling mammalian skin. However, unlike mammals’ and terrestrial vertebrates’ epidermis, which is covered by an outer layer of keratinized dead cells, zebrafish skin surface is made of living cells that are covered with mucus and lacks a cornified envelope [ 21 ]. Furthermore, zebrafish has no mammalian appendages, since hair follicles and sebaceous glands cannot be detected. However, zebrafish presents the breeding tubercle, which is an epidermal appendage shared with mammals [ 22 ].

Mammalian epidermis is a well-organized stratified tissue that includes basal, spinous, granular, and horny cells from the basal membrane to the skin surface. Teleost epidermis only has three layers [ 23 ]. The surface layer is a single cell layer in which cells are rich in keratin filaments and are continuously replaced at their death, without producing a stratum corneum. The intermediate layer is composed of different cell types, including unicellular glands (mucous cells and club cells), sensory cells, ionocytes and undifferentiated cells. The basal layer is a single cell layer which is attached to the basement membrane via hemidesmosomes, which tightly link the epidermis to the dermis [ 24 ].

Maturation of zebrafish from embryo to fully developed fish only takes a few days. Layers representing the epidermis and the dermis are already detectable at one day post-fertilization (dpf). In adult zebrafish, scales covering the epidermis form at around the 30th dpf and sonic hedgehog pathway has been identified has having a role in their development [ 25 ]. Collagenous stroma formation is dependent on fibroblasts, whereas pigment production derives from melanocytes, belonging to neural crest-derived pigment cell system [ 26 ].

Several epidermal marker genes, including keratins 1 and 5, the 230 kDa bullous pemphigoid antigen, plectin, and several cutaneous basement membrane zone (BMZ) genes, including type IV, VII and XVII collagen, are expressed in zebrafish skin in early developmental stages. Most expressed human collagens types, including collagens I, V, and VI, are detectable in zebrafish skin from 6th dpf. In conclusion, zebrafish repertoire of genes involved in cutaneous development reveals strong similarities with human skin [ 25 ].

The zebrafish neural crest produces three different kinds of pigment cells: melanophores, xanthophores and iridophores (Fig.  1 ). Melanophores synthesize melanin and are analogous to melanocytes of vertebrates, xanthophores have a yellow appearance caused by pteridine pigments, and iridophores contain iridescent platelets which reflect light. Melanophores firstly develop among pigmented cells at approximately 24 h post fertilization from melanogenic progenitors deriving from the neural crest [ 27 ].

Comparison between zebrafish and human skin. Both zebrafish and human adult skin include a multi-layered epidermis, a basal membrane (bm) and an underling dermis containing collagen (col) fibers. Zebrafish epidermis contains mucous cells (muc), while human epidermis has a stratum corneum (cor) as outermost layer. Zebrafish pigment cells include xanthophores (xp), iridophores (ip) and melanophores (mp); human pigment cells are represented by melanocytes (mc). Images created with BioRender.com

Like in mammalian melanocytes, the tyrosine-protein kinase KIT has a major role in promoting the initial migration of melanocytes in the first two days of the embryos [ 28 ]. Later in zebrafish larval development, a new set of melanocytes contribute to formation of stripes characterizing adult zebrafish after metamorphosis [ 29 ].

2.2 Skin inflammation in zebrafish

Skin is essential in defending fish from environmental stress factors. Since fish are poikilotherms, even small changes in the external parameters may lead to injury and inflammation [ 30 ]. A series of epithelial cells, resident non-immune cells, vascular endothelial cells and mucosal epithelial cells help initiate and coordinate the inflammatory response [ 31 , 32 , 33 ]. Interestingly, unlike humans, [ 34 ]. fish do not have major lymphoid accumulations. It is still unclear where fish lymphoid cells naturally reside. The most probable theory is that fish leukocytes migrate to the skin via mucus secretions in response to damage stimuli [ 35 , 36 , 37 ].

Inflammation pathways are regulated by the NF-κB family transcription factor both in mammals and fish [ 38 ] and classic pro-inflammatory cytokines such as IL-1β, TNF-α and IL-6 prevail as paralogues in most teleosts [ 39 , 40 , 41 ], thus making the main mechanisms of inflammation similar in the two species. Neutrophils play an essential role in initiating the inflammatory response in both mammals and zebrafish and in perpetration of the inflammation, especially via TNF-α and IL-1β [ 42 , 43 , 44 , 45 , 46 , 47 , 48 ].

Once activated, monocytes differentiate into classically pro-inflammatory macrophages, functioning as antigen presentation cells and producing reactive oxygen species (ROS), TNF-α and IL-1β [ 42 , 49 , 50 , 51 , 52 ].

Besides, neutrophils activation determines exocytosis of their granules [ 42 , 50 , 51 , 53 , 54 ]. Antigen presenting cells such as dendritic cells, macrophages and endothelial cells are also recruited by neutrophils [ 51 ]. While in mammals leukocytes originate in the bone marrow and mature in lymph nodes, zebrafish lacks such structures [ 55 ]. Specifically, the bone marrow has its counterpart in the head kidney acting as a major hematopoietic and lymphoid organ. Indeed, the thymus, spleen and mucosa-associated lymphoid tissues (MALT) are shared between fish and mammals [ 56 ]. Migration and proliferation of the immune cells in zebrafish skin has been recently studied using fluorescent light which induced the early expression of skin genes associated with inflammation [ 57 ].

3 Zebrafish as a model system in oncology

One of the biggest obstacles in the field of oncology is to address cancer heterogeneity either in inter-individual differences or in intra-tumoral contexts [ 58 , 59 ]. Innovations in cancer research have largely benefited from further exploring these processes in live animals, with a strive to identify and target the most frequent driver mutations as a rational approach to treatment [ 60 ]. This is especially done in early tumor development at a cellular level.

As an example, patient-derived cancer cell xenotransplantation (PDX) could help to overcome treatment resistance due to added mutations in tumor cells, by means of large-scale drug (small molecules) screening. This process is not as easily achievable in murine models as it may be in non-mammalian models such as fish, since recipient immunosuppression is required for PDX in mice [ 61 , 62 , 63 ].

The zebrafish has recently caught attention due to the aforementioned characteristics, alongside its highly-conserved cancer signaling pathways compared to the human species.

Moreover, the zebrafish is a versatile model, as it is possible to operate a mutation in a specific gene, thus creating a stable transgene, or to create a transient over-expression or down-regulation of a specific gene. Forward and reverse genetic screens are also possible in zebrafish [ 64 ].

Initially, zebrafish have been used in forward genetic screens to test the effects of mutagens on neoplasm development. Ethylnitrosurea screens leading to mutations in tp53, one of most studied genes among those involved in cancer pathogenesis, were among the first experiments in zebrafish in the field of oncology [ 65 , 66 ].

Further techniques have been subsequently introduced, which helped cancer studies in zebrafish to progress. The aim was to create loss-of-function phenotypes or to introduce transgenes that are typically mutated in human cancer into fish models. Research in this field brought forward evidence that many mutated tumor suppressor genes, such as Tp53, and oncogenes, such as mMyc and KRas, could generate parallel tumors in zebrafish in the same way as they had been observed in humans. This shed light on the evolutionary conservation of drivers and pathways of tumorigenesis between man and fish [ 67 ].

Currently, the most used techniques for gene manipulation in zebrafish are morpholino oligomers (MOs) [ 68 ], zinc finger nucleases (ZFNs) [ 69 ], transcription activator-like effector nucleases (TALENs) [ 70 ] and the CRISPR (clustered regularly interspaced short palindromic repeats) system [ 71 ]. However, new promising techniques have recently been introduced to be used in zebrafish, such as TEAZ (transgene electroporation in adult zebrafish) and tumor cell transplantation, especially in the form of PDX (patient-derived cancer cell xenotransplantation).

MOs are small synthetic oligomers that block mRNA translation in vivo; they are easy to use and enable us to obtain models in a short amount of time, despite concerns about their off-target effects and their inexact reproduction of genome-editing mutants, thus requiring a control with the knockout phenotype [ 68 , 72 , 73 ].

ZNFs is a useful technique for multiplex gene targeting to be performed in one round, either creating knock-outs (loss of function) or knock-ins (gain of function) [ 69 ].

TALENs enable us to produce heritable gene disruptions in the vertebrate genome; more importantly, they can create mutations in somatic tissues with a high success rate, including bi-allelic mutations [ 70 ].

CRISPR/Cas9 is a technique in which Cas9 endonuclease recognizes a specific DNA sequence by means of a guide RNA sequence binding both DNA and Cas9. Zebrafish models based on this technique are widely used today due to the potential possibility to target multiple genes at the same time and to its high efficiency [ 71 , 74 , 75 ].

TEAZ is a new technique that enables the injection of DNA constructs containing tissue-specific promoters and genes of interest into adult tissue. In addition, TEAZ is extremely fast as far as tumor onset is concerned, and the expression of genes of interest can be evaluated in adult fish [ 76 ]. TEAZ is very promising compared to conventional zebrafish cancer models created by means of the aforementioned techniques. In fact, the latter involves the injection of nucleic acids into one-cell stage embryos. Therefore, it is sometimes difficult to study cancer pathogenesis and development in animal models, since the onset and the site of the developing tumors are not accurate, and the spreading of metastases could be hard to evaluate. Alongside TEAZ, cancer cell transplantation in zebrafish embryos and adults could partially overcome the problems connected with common techniques [ 77 ].

Tumor cell transplantation is an important tool in studying tumor invasiveness. It involves cancer cell transplantation from a donor to a recipient of the same species (allograft) or of a different species (xenograft) [ 78 ].

Many studies have demonstrated that zebrafish embryos can engraft human cancer cells and give precious insight into disease pathogenesis.

As for human cancer xenotransplantation, zebrafish have some advantages compared to murine models, especially because a high number of transparent embryos lacking a mature immune system can be transplanted with cancer cells and tracked. In other words, visualization of cell-cell interactions in vivo is possible in zebrafish. Moreover, PDX in zebrafish can help us find new targets for targeted anti-cancer treatments. There is evidence that pre-clinical research might shorten the time for drug approval, mostly due to drug re-purposing [ 10 ]. The zebrafish has already shown to be a reliable model to assess drug efficacy and sensitivity, since in some experiments patient-derived cells responded well to the same drugs that were used in patients [ 79 ].

Thus, the use of zebrafish as a pre-clinical screening model for patient-derived cancer cell xenotransplantation might revolutionize our approach to cancer, especially in a personalized medicine perspective, and explains the growing interest in PDX studies in zebrafish [ 77 ].

The variety of cancer types that have been successfully reproduced in zebrafish prove that this animal model has a lot of potential in the analysis of almost every type of cancer observed in humans. Genetic models of cancer in zebrafish include peripheral nerve sheath tumor (PNST) [ 80 , 81 , 82 ], rhabdomyosarcoma (RMS) [ 83 , 84 ], melanoma, [ 85 , 86 , 87 , 88 , 89 , 90 ] thyroid cancer [ 91 ], pancreatic cancer [ 92 , 93 ], hepatocellular carcinoma (HCC) [ 94 , 95 , 96 ], intestinal tumors [ 97 , 98 ], testicular tumors [ 99 ], T-cell acute lymphoid leukemia (T-ALL) [ 83 , 100 , 101 , 102 ], Acute Lymphoid leukemia (AML) [ 103 , 104 , 105 , 106 ], chronic myeloid leukemia (CML) [ 102 , 107 ], myelodysplastic syndrome (MDS) [ 108 ].

Some of these cancer types, along with others, have been studied with PDX in Danio rerio [ 10 ]. Interestingly, the zebrafish has proved to be a reliable model for PDX for some cancers that develop in human organs that fish do not have, such as the breast, prostate and lungs [ 109 , 110 ]. It has proved to be a good model for studying rare cancer pathogenesis as well, such as Ewing sarcoma [ 111 ].

3.1 Melanoma models in zebrafish

To better understand the mechanisms underlying melanoma, the zebrafish represents an excellent model through the use of xenografts [ 112 ] and transgenic models [ 113 , 114 ].

Melanoma has certainly been one of the most studied cancers and the most analyzed skin cancer in zebrafish, since the first description of BRAF V600E model. It is known that the V600E mutation, a key melanoma driver found in about 43–50% of melanomas [ 10 , 115 , 116 ], is also frequently found in benign naevi and moles which do not progress to cancer. It is also known that the loss of function of the tumor suppressor gene p53 (p53−/−) is required for cancer progression in naevi . However, the long time lapse and rarity of melanoma tumor formation (one to three in a fish’ lifetime) in zebrafish carrying both BRAF V600E and p53−/− mutations, imply that there are other molecular alterations and pathways playing a role in melanoma formation. Based on the observation that crestin, the expression of which is generally limited to neural crest progenitor cells in developing zebrafish embryos, was expressed in zebrafish melanomas [ 117 ], studies were performed in which engineered transgenic zebrafish expressing GFP (green fluorescent protein) under the control of crestin-regulatory elements were tracked. GFP-positive cells showed that only individual melanocytes that reactivated crestin could initiate melanomas. This highlighted that melanoma at a one-cell-state is based on reprogramming the cell to become more neural-crest-like [ 86 ]. As confirmation, consistent with crestin expression was the expression of the SOX10 transcription factor, a conserved early neural crest marker that helps melanocyte reprogramming to an embryonic state. Reactivation of neural crest genes such as crestin (in zebrafish melanoma) and SOX10 (in zebrafish as well as in human melanoma cell lines) is probably consequent to epigenetic modifications on histones, as shown by some histone markers known as super-enhancers [ 117 ].

N-RAS mutation has also been studied in zebrafish and its expression led to hyperpigmentation throughout the zebrafish’s body. When p53 mutation was added to mutated N-RAS, the fish developed invasive melanomas which were histologically and genetically correlated to human melanomas.

Although less frequent in melanomas than the previously mentioned BRAF and N-RAS mutation, H-RAS-mutated zebrafish models also displayed melanoma development [ 90 , 118 , 119 ].

Combining these assets with the excellent melanoma models engineered in zebrafish has led to several significant advances in our knowledge of melanoma behavior and molecular asset. New frontiers involve testing even infrequently mutated potential drivers, thus broadening the available models of cutaneous melanoma and introducing non-cutaneous melanoma zebrafish models [ 120 ]. Moreover, loss of function CRISPR/Cas9 gene targeting technology has been successfully used to create loss of function models, allowing testing of candidates that may alter disease onset and/or progression.

As an example, this technique was used to investigate SPRED1 function as a tumor suppressor in the context of KIT mutations in mucosal melanoma. SPRED1 knockdown, determining MAPK activation, conferred resistance to drugs inhibiting KIT tyrosine kinase activity. MAPK inhibition in SPRED1-deficient melanomas could therefore be a therapeutic hint and again proves the power of zebrafish modeling to investigate genetic interactions in cancer pathways [ 121 ].

Concerning the aforementioned cancer intra-tumoral heterogeneity, single cell RNA sequencing (sc-RNA sq) technologies provide an insight into melanoma complexity [ 122 ]. Analysis of cell dynamics at the minimal residual disease (MRD) stage, when persistent cells in otherwise disease-free tissue acquire specific properties for melanoma progression, proves fundamental to grasp the tumor vulnerability at a crucial point [ 123 ]. Sc-RNA sq was used to study MITF-low state role in melanoma progression in zebrafish genetic models with low activity of Mitfa, proving that very low or absent MITF activity characterized a residual disease like therapy-resistant melanoma [ 124 ]. Additional research on melanoma cells interaction with their microenvironment has been accomplished in a transgenic zebrafish model, proving the power of tools such as spatially resolved transcriptomics, sc- RNA-seq, and single-nucleus RNA-seq [ 125 ].

Interaction with metabolism has rarely been considered as an impacting factor in cancer and, more specifically, in melanoma; however, interference with liver gluconeogenesis has been successfully investigated in a zebrafish melanoma model through isotope tracing, confirming versatility of zebrafish in the field of research [ 126 ].

Not only has the zebrafish model helped to investigate melanoma genesis and development as far as its genetics is concerned, but also it has recently offered an insight into new therapeutic strategies for melanoma metastatic progression by targeting specific signaling cascades. For instance, human epidermal growth factor receptor (EGFR) signaling was implied when PLD c GMP analog protein kinase G activator 5 (PA5) was injected into zebrafish melanoma models, thus targeting the cGMP/protein kinase G pathway [ 127 ]. Another receptor tyrosine kinase, Xrmk, was identified as closely related to EGFR, and therefore involved in melanoma development and progression; in detail, Xrmk has been studied in Xiphophorus platyfish and in zebrafish as a therapeutic target [ 128 ]. Moreover, the activation of CD271, a member of the tumor necrosis factor receptor (TNFR) family, using a short β-amyloid-derived peptide, combined with chemotherapy or MAPK inhibitors, proved to significantly reduce metastasis in a zebrafish xenograft model [ 129 ].

3.2 Squamous cell carcinoma models in zebrafish

Even though non-melanoma skin cancer in fish is less common compared to melanoma, zebrafish have been adequately used as a model to study the underlying pathogenetic mechanisms in these kinds of cancer as well.

Recent works that employ the SCC xenograft model in zebrafish have identified key molecules involved in the pathogenesis of squamous cell carcinoma (SCC) [ 130 ], as well as compounds that may be used as targets for SCC therapy [ 131 ]. A crucial molecule to be studied as a therapeutic target is the tyrosine kinase receptor Axl, which is highly expressed in SCC [ 132 ]. Other important targets are the COL7A1 gene, which is responsible for the development of aggressive SCCs in epidermolysis bullosa, and the recombinant type VII collagen (hrCol7), which is able to reverse SCC angiogenesis in the zebrafish model [ 133 ].

Another interesting in vivo xenograft model study has analyzed the role of the tyrosine kinase discoidin domain receptor 2 (DDR2) in cell proliferation, adhesion, differentiation and invasion in head and neck squamous cell carcinoma (HNSCC) [ 134 ]. The study shows that dasatinib, a Food and Drug Administration (FDA)-approved inhibitor of c-Kit, Proto-oncogene tyrosine-protein kinase (ABL, SRC) and Abelson murine leukemia viral oncogene homolog, may be potentially used in DDR2-positive SCC patients to block tumor cell invasion and migration [ 134 ].

Another potential compound for HNSCC treatment is the marine microbial extract luminacin. Studies in zebrafish embryos have shown that luminacin treatment of tumor cells stimulates autophagy in SCC cell lines, thus inhibiting cancer growth and progression [ 130 ].

Lastly, the zebrafish model has also been used to show that Flotillin-1 over-expression in KB cells (a subline of the keratin-forming tumor cell line HeLa) boosts KB cell motility and cell growth [ 135 ].

These studies prove that the zebrafish model may be adequately used not only in the evaluation of molecular pathways involved in SCC development and progression, but also in drug toxicity and screening assays.

3.3 Other dermatological applications of zebrafish

Zebrafish can be used to study not only cancer derived from melanocytes, but also other disorders of melanogenesis, since melanogenesis pathways are conserved between zebrafish and mammals, and melanogenesis is a visible process in zebrafish embryos and in transparent casper adults [ 136 ]. Studying zebrafish albinism models, researchers could clarify the function of genes whose role in the pathogenesis of this disorder remains concealed and that might not yet be recognized as implicated in human albinism. Correct genetic diagnosis might prove crucial in treatment of different, but often clinically indistinguishable, forms of albinism. Thus, rapid CRISPR screening for gene function makes zebrafish an excellent model for albinism gene discovery. Though counterintuitive, zebrafish albinism models could also help clarify chemotherapeutic resistance mechanisms in cancerous melanocytes in melanoma [ 137 , 138 ].

Hereditary pigment disorders have been investigated using MOs to ascertain the function of specific genes that had previously been identified in affected individuals. Protein O-fucosyltransferase 1 (pofut1) and presenilin enhancer-2 (psenen) knockdown zebrafish both displayed abnormal distribution in pigmentation, thus confirming involvement of the aforementioned genes in certain clinical presentations of Dowling-Degos syndrome, also known as reticulate pigmented anomaly of flexures. Furthermore, oca2 -mutant zebrafish and c10orf11 knockout zebrafish were created to explore oculocutaneous albinism-related gene function in vivo, confirming involved conserved gene function throughout fish, mouse and humans. Hypopigmentation characterized also snow white zebrafish mutant carrying a hps5 gene mutation, reproducing Hermansky-Pudlak syndrome (HPS) in fish models. Zebrafish fade out mutant also recreated HPS phenotype indicating that fade out gene could have a role in the pathogenesis of HPS. Disorders of copper metabolism were also reproduced in zebrafish with the calamity and catastrophe mutant models, underlying the influence that copper and, potentially, other nutrients, could have on melanin synthesis in melanocytes. Impact of stress on vitiligo development in fish was reproduced by treating zebrafish with interleukin-17, which determined altered pigmentation and autophagy in pigment cells [ 138 ].

Mutation in NRAS resulting in an I24N amino acid substitution was identified in an individual bearing typical Noonan syndrome features. N-Ras-I24N expressing zebrafish displayed developmental defects which were parallel to other Noonan syndrome-associated genes in zebrafish. Activation in N-RAS signaling pathway was therefore confirmed to be associated to a Noonan Syndrome phenotype. Of note, MEK inhibition completely rescued the activated N-Ras-induced phenotypes, confirming the exclusive mediation of Ras-MAPK signaling in the genesis of the syndrome [ 139 ].

Co-occurrence of Mongolian blue spots with vascular birthmarks defines a group of syndromes known as phakomatosis pigmentovascularis. Association with activating mutations in GNA11 and GNAQ genes, encoding a Ga subunit of heterotrimeric G proteins, was discovered and confirmed in a transgenic mosaic zebrafish model expressing mutant GNA11R183C under mitfa promoter, which developed extensive dermal melanocytosis recapitulating the human phenotype. Specifically, zebrafish embryos were injected with wild type human GNA11, GNA11R183C, or GNA11Q209L expressed under control of the melanocyte mitfa promoter. The embryos were grown to adult fish; their status of genetically mosaic animals was clinically visible as melanocyte patches, which received histological confirmation [ 140 ].

RASopathies result from germline mutations of the Ras/MAPK pathway. Systematic predictions on disease progression are not yet possible, even though available technologies in genome sequencing allow to identify multiple disease-related mutations. Nevertheless, zebrafish embryos represent a valuable model in assessing mutational effects. Jindal et al. succeeded in ranking several MEK1 mutations, proving that those found in cancer were more severe than those found in shared by RASopaties and cancer. Also, the latter resulted as more severe than those characterizing only RASopaties. A conserved ranking was observed in Drosophilaand the ranking could predict the drug dose to correct the defects [ 141 ]. Wound healing and re-epithelialization of adult zebrafish skin have been analyzed in several studies. In zebrafish the process of wound healing results in minimal scar formation. The process comprises a series of events: rapid re-epithelialization; migration of inflammatory cells; formation of granulation tissue consisting of macrophages, fibroblasts, blood vessels, and collagen; granulation tissue regression. Major steps and principles of cutaneous wound healing seem to be the same in adult mammals and adult zebrafish, thus making the zebrafish a valuable model for studying vertebrate skin repair [ 142 ]. Richardson et al. studied the wound healing process by creating full-thickness wounds with a laser on the flank of adult zebrafish in a rapid and reproducible way, confirming that the zebrafish is a unique and cost-effective model for skin repair [ 142 ]. Absence of wound scars in zebrafish, as observed in human embryos, due to the lack of the blood-clotting phase and to specific signaling mechanism, represents an attractive model to study healing processes and is expected to help to formulate an appropriate drug for cutaneous wound healing [ 143 ].

The aforementioned similarities of the zebrafish integument structure together with those of the inflammation mechanisms, make this teleost a fundamental and cost-effective model also to study major dermatologic inflammatory diseases, such as psoriasis. Several models, including mutant, morphant and environmentally inducible models, were created to investigate genetic alterations and molecular mechanisms of psoriasis [ 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 ].

4 Conclusions

Inflammatory and neoplastic skin disorders are very common and are increasing worldwide.

Zebrafish can provide a suitable animal model to extend our understanding of the molecular and cellular mechanisms of skin disorders and to develop new therapeutic strategies in dermatology (Fig.  2 ). Zebrafish models of major interest in dermatological research are summarized in Table  1 [ 22 , 39 , 85 , 86 , 87 , 89 , 90 , 147 , 153 , 154 , 155 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 ].

Zebrafish applications in skin biology. Examples of applications of the zebrafish model in the field of skin biology include skin disease and tumor modeling, biochemical and genetic tests, drug screen and in vivo imaging, all suitable for large-scale studies. Images created with BioRender.com.

Owing to its low maintenance cost, highly conserved genome, and easy genetic manipulation, the zebrafish is an excellent model for preclinical research in dermatological laboratories, thus bridging the gap between in vitro cell culture an in vivo mammalian models.

5 Methodological approach

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Acknowledgements

NT was supported by AIRC (Grant IG-2017-19928), Italian Telethon (Grant GGP19287), IOV 5 × 1000 (Gant METAMELAHP-NAP), and “Piccoli Punti” foundation.

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Irene Russo, Emma Sartor and Laura Fagotto contributed equally to the work

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Irene Russo, Emma Sartor, Laura Fagotto, Anna Colombo & Mauro Alaibac

Department of Biology, University of Padua, Via U. Bassi 58/B, 35131, Padua, Italy

Natascia Tiso

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Russo, I., Sartor, E., Fagotto, L. et al. The Zebrafish model in dermatology: an update for clinicians. Discov Onc 13 , 48 (2022). https://doi.org/10.1007/s12672-022-00511-3

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Zebrafish in Toxicology and Environmental Health

As manufacturing processes and development of new synthetic compounds increase to keep pace with the expanding global demand, environmental health, and the effects of toxicant exposure are emerging as critical public health concerns. Additionally, chemicals that naturally occur in the environment, such as metals, have profound effects on human and animal health. Many of these compounds are in the news: lead, arsenic, and endocrine disruptors such as bisphenol A have all been widely publicized as causing disease or damage to humans and wildlife in recent years. Despite the widespread appreciation that environmental toxins can be harmful, there is limited understanding of how many toxins cause disease. Zebrafish are at the forefront of toxicology research; this system has been widely used as a tool to detect toxins in water samples and to investigate the mechanisms of action of environmental toxins and their related diseases. The benefits of zebrafish for studying vertebrate development are equally useful for studying teratogens. Here, we review how zebrafish are being used both to detect the presence of some toxins as well as to identify how environmental exposures affect human health and disease. We focus on areas where zebrafish have been most effectively used in ecotoxicology and in environmental health, including investigation of exposures to endocrine disruptors, industrial waste byproducts, and arsenic.

1. INTRODUCTION

Rapid growth of populations and technological advancement has resulted in innumerable pollutants and environmental toxin exposure. This has generated a vital need for toxin surveillance, identification of consequences of exposure, and understanding of the biologic, chemical, and genetic mechanisms that underlie those effects ( Landrigan, 2016 ). The field of environmental health was established as early as the 1940s, in response to the expansion of chemical manufacturing and the occurrence of contamination of the water, soil, and air caused by widespread use of chemicals in industry and consumer products ( Landrigan, 2016 ). Since World War II, thousands of synthetic chemical compounds have been created for industrial applications and have subsequently been introduced into consumer products. Today, approximately 70,000 chemicals are in commercial use in the United States, and 3300 of these are high production volume compounds, with annual production or importation volumes in excess of one million pounds. In addition to the risks posed by the expanding repertoire of manufactured toxins, naturally occurring chemicals, such as metals can also cause harm, as was recently brought to focus by the lead contamination of the drinking water in Flint, Michigan ( Bellinger, 2016 ; Tong, Baghurst, McMichael, Sawyer, & Mudge, 1996 ).

Environmental toxins profoundly affect fish and wildlife. In particular, water pollution has damaged fish populations by affecting reproductive health, lifespan, and embryonic and larval development. This has a major effect on aquatic ecosystems and on the industries that depend on them. Humans are exposed to environmental toxicants through fine particulate matter in the air, endocrine-disrupting chemicals (EDCs) found in food packaging, household items and personal care products, and naturally occurring compounds such as metals. Human exposure to environmental chemicals is associated with both acute toxicity and long-term consequences ( Landrigan et al., 2016 ), which include congenital abnormalities ( Swan et al., 2005 ), chronic diseases ( Argos et al., 2010 ; Mazumder, 2005 ), cognitive disabilities ( Jacobson, Muckle, Ayotte, Dewailly, & Jacobson, 2015 ; Muñoz-Quezada et al., 2013 ; Tong et al., 1996 ), cancer ( Liu & Wu, 2010 ; Selikoff & Hammond, 1968 ; Wang, Cheng, & Zhang, 2014 ), and death ( Argos et al., 2010 ). The field of environmental health is expanding to meet the demands of surveillance and prevention of consequences of environmental toxin exposure on both wildlife and human health.

There are many unanswered questions in the field of environmental health ( Henn, Coull, & Wright, 2014 ; Landrigan, Suk, & Amler, 1999 ), and a surge in research effort is required to answer these. Among the most pressing are: What are the effects of low dose, cumulative exposures, and exposures to multiple toxicants? What are the developmental processes that are altered by toxicant exposure and how are these processes affected? What are the latent effects of early life exposure? Are these effects apparent in subsequent generations? Can we develop surveillance technologies to limit exposure? How can therapeutic interventions be designed and administered to reverse the effects of exposure? The barriers to addressing these questions in human populations are both practical and logistical. In terms of low-dose and cumulative exposures, the appropriate biomarkers and the ideal tissue specimens for analysis have not been identified for every toxicant or combination of toxicants. Understanding the latent and transgenerational effects of exposure is difficult in humans due out long lifespans and relatively small number of offspring. In addition, the interaction between environmental toxicants and social “exposures” including chronic stress, exposure to violence, and nutrient scarcity are only beginning to be understood. There is a critical need for in vivo animal models to study the short- and long-term effects of environmental toxins.

Zebrafish are a valuable tool for Environmental Health researchers as evidenced by a rapidly expanding body of research using zebrafish. A PubMed search using the terms “zebrafish environmental health” reveals that the use of zebrafish in this field has been steadily increasing over the past few decades ( Fig. 1 ). In this chapter, we will highlight the unique advantages of using zebrafish embryos, larvae, and adults to address pressing issues in Environmental Health, including contaminant detection, environmental monitoring, toxicity/teratogenicity testing, and investigations into mechanisms of action and disease phenotypes associated with exposure to chemical compounds. In a field that rapidly changes with evolving technology and manufacturing worldwide, zebrafish can offer real-time in vivo studies to address potential hazards to human health that result from naturally occurring compounds and commercial use of new synthetics or byproducts of their production, and can improve our limited understanding of the specific effects of environmental exposures.

Increasing use of zebrafish in environmental health studies. The use of zebrafish has steadily increased during the past three decades. Line graph represents the number of Pubmed articles categorized under “zebrafish environmental health.” Dashed arrows indicate government programs and legislation focused on toxicology and environmental health from 1975 to present.

2. HISTORICAL PERSPECTIVE: TOXICOLOGY AND GOVERNMENT EFFORTS FOR ENVIRONMENTAL REGULATION

In 1976, the United States Congress passed the Toxic Substances Control Act, which granted the Environmental Protection Agency (EPA) the authority to require testing and reporting, and set restrictions on the manufacture and use of chemicals and mixtures ( Toxic Substances Control Act, 1976 ). Since the passage of this law, science, and technology have made rapid progress necessitating further legislative intervention to protect the public from the health effects of chemical exposures ( Birnbaum, 2010 ). In response, the National Toxicology Program was established in 1978 as an interagency program by the Department of Health, Education, and Welfare, which is known today as the Department of Health and Human Services, to address public, scientific, and governmental concerns that human diseases and disabilities are linked to chemical exposures ( Xie, Holmgren, Andrews, & Wolfe, 2016 ). In 1980, as part of the Comprehensive Environmental Response, Compensation and Liability Act, more commonly referred to as the “Superfund Act,” Congress formed the Agency for Toxic Substances and Disease Registry (ATSDR). ATSDR is a science-based public health agency created to study the health effects of hazardous substances in the environment and to work with communities to keep them safe from hazardous waste. To address this mission, ATSDR collects data and conducts studies in addition to using the best available scientific data to make recommendations to the EPA and other agencies to prevent and stop exposures to ensure the health of communities. Studies in zebrafish have provided critical information about the health effects of a majority of the 250+ substances on this list. Based on the strength of published data and the size of potentially exposed population, the ATSDR publishes a biannual Priority List of Hazardous Substances to select those substances that should be the subject of toxicological profiles.

With the dissolution of the National Children’s Study 2014, the NIH reappropriated funds to create the Children’s Health Exposure Analysis Resource (CHEAR) to focus on how children’s health is shaped by the environment. Through CHEAR, researchers with NIH-funded child cohorts can apply to have biological samples analyzed for chemicals, metabolites, and biomarkers of exposure. In 2016, President Obama signed the Frank R. Lautenberg Chemical Safety of the 21st Century Act into law requiring that the EPA perform testing of chemicals currently in use, set new risk-based safety standards, require protection for vulnerable populations, and increase public transparency for chemical information. The use of zebrafish has and will continue to be a tool to provide the EPA with invaluable information regarding the short-term toxicity and long-term health effects of toxicant exposure to human health and its utility has been highlighted in a special issue of the journal Zebrafish that was dedicated to the use of this model in toxicology research ( Gamse & Gorelick, 2016 ).

3. ENVIRONMENTAL AND DEVELOPMENTAL TOXICOLOGY

Zebrafish are commonly used to model human diseases using genetic modifications; applications including studies of heart, kidney, liver, hematopoietic, immune, and other systems detailed other chapters in this volume. They can similarly be used to model the health effects of environmental exposures to better understand the etiologies and mechanisms of environment-related disease in humans. The concern is growing over the persistence of chemical compounds in the environment as well as the acute and long-term health effects of exposure to environmental toxicants and contaminants and zebrafish provide an ideal model to study these effects. Chemicals can simply be added to the embryo medium and the developing and transparent zebrafish can be assessed for lethality and developmental abnormalities from fertilization through larval stages. Although juvenile and adult zebrafish are not transparent, the generation of the unpigmented Casper mutant line can be crossed to transgenic fluorescent reporters to aid observation and imaging of organ systems in older zebrafish ( White et al., 2008 ).

The ability to observe effects of toxins in vivo allows for direct assessment of toxicity, as well as measurements of absorption, distribution, metabolism, and elimination. This can be extended for use in screening for treatments that can mitigate toxic effects in live animals as well. Zebrafish express a full range of cytochrome P450 ( cyp ) genes required for xenobiotic metabolism and biotransformation ( Goldstone et al., 2010 ). In the zebrafish genome assembly (GRCz10), a total of 86 cyp genes were identified ( Saad et al., 2016 ) with many of the metabolic characteristics of the related human enzymes, demonstrating a strong evolutionary relationship with those found in humans. However, there remains a significant lack of information about the specific mechanisms of zebrafish xenobiotic Cyp activity.

Zebrafish have been used to study the compounds ranging from naturally occurring metals and metalloids, to synthetic components of consumer products, pesticides, and byproducts of industrial processing and waste incineration. In this chapter, we will present the zebrafish tools that have been employed for the detection of these toxicants and how zebrafish research is contributing to the understanding of the effects of these compounds on the environment and on human health.

4. ZEBRAFISH: TESTING THE WATERS FOR TOXICANTS

In 1982, George Streisinger, the founder of the zebrafish field ( Streisinger, Walker, Dower, Knauber, & Singer, 1981 ), proposed the use of zebrafish as a vertebrate model to study the frequency of mutations in response to environmental carcinogens ( Streisinger, 1983 ). In the following three decades, zebrafish have been used to identify teratogens, to uncover mechanisms of action of common toxicants, and to understand the tissue specificity of toxicant impact on vertebrates ( Gamse & Gorelick, 2016 ). Zebrafish provides a unique, in vivo, medium-throughput system to expand cell culture assays to a whole vertebrate model, but are less expensive than rodents. The benefit of the large population size of zebrafish offspring is a major benefit, as studies in zebrafish allow for the rapid assessment of compound toxicity and the ability to study molecular mechanisms underlying developmental and health outcomes associated with toxicant exposure across a population of live vertebrates. In addition, large numbers of offspring enable longitudinal studies that can be done on a population scale of the developmental effects of environmental exposures at a relatively less cost than longitudinal rodent studies. Zebrafish are also easily amenable to drug discovery screens as sentinels of environmental contamination, for toxicity testing, and for investigations into the mechanisms of action of pharmaceuticals and toxicants. The zebrafish model provides the opportunity to combine the power of rapid toxicology screens with the ability to study the association of exposures with long-term outcomes in a vertebrate, making zebrafish an invaluable complementary system for research in Environmental Health.

4.1 Transgenic Zebrafish as Surveillance Tools

For nearly two decades, zebrafish have been used for biomonitoring. A major advantage of using zebrafish for this work is that the embryos and larvae are transparent and generating transgenic animals is relatively easy. This has allowed the development of transgenic lines where a fluorescent protein or other measureable readout becomes activated in the presence of contaminants or environmental stressors provide a system to assess the level of response and the tissue specificity of the response ( Carvan, Dalton, Stuart, & Nebert, 2000 ; Gorelick, Iwanowicz, Hung, Blazer, & Halpern, 2014 ; Lee, Green, & Tyler, 2015 ). In the earliest efforts, investigators developed transgenic zebrafish lines in which expression of the luciferase or green fluorescent protein ( GFP ) gene is driven by pollutant response elements that report on the presence of aromatic hydrocarbons, electrophiles/oxidants, metals, estrogenic compounds, or retinoids ( Carvan et al., 2000 ). Transgenic lines have been used not only to detect the presence of toxins, but can facilitate investigations into the molecular mechanisms underlying pathology associated with environmental exposures. The use of transgenic reporter zebrafish lines to measure exposure to heavy metals, organic chemicals, endocrine disruptors, and electrophilic agents has been expertly reviewed elsewhere ( Lee et al., 2015 ) and are outlined in Table 1 . We describe how such tools are used to both detect the presence of contaminants and to understand their physiological impact.

Transgenic Zebrafish Lines for Reporting Toxicant Exposure

4.2 Biosensors of Environmental Contaminants

Zebrafish have been used as sentinels to identify the effects of public water supplies. An early study examined the teratogenic effects of sediment and ground water in the Netherlands in zebrafish combined with a biochemical assay in tissue culture cells and found that the zebrafish teratogen assay was equally as sensitive in identifying the presence of toxic contaminants ( Murk et al., 1996 ). In more recent work, several researchers have generated transgenic reporter lines in which a promoter drives expression of a fluorescent protein or other reporter that is regulated by exposure to a toxin ( Table 1 ). In this section, we will highlight specific transgenic lines that have been generated and used to not only identify classes of chemical contaminants in experimental settings, but with the potential to lend insights into toxicant-induced stress responses.

4.2.1 Aromatic Hydrocarbons and the Aryl Hydrocarbon Receptor (Ahr)

The aryl hydrocarbon receptor (Ahr) is a cytosolic receptor that is expressed in various tissues during development and adulthood, and signaling through this receptor has been studied in multiple developmental processes in rodents and zebrafish ( Schneider, Branam, & Peterson, 2014 ). The Ahr is activated in response to synthetic and natural aromatic (aryl) hydrocarbons and functions as a transcription factor to bind to the dioxin-responsive element (DRE) to induce the expression of genes including those encoding the CYP enzymes, which are involved in xenobiotic metabolism. A DRE-containing fragment of the cyp1a1 gene, which is regulated by Ahr, was used to drive expression of a nuclear-localized GFP ( Tg(cyp1a:nls-gfp) ) and this shows activation in response to 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) ( Kim et al., 2013 ). However, more recently, the Tg(cyp1a:gfp) transgenic line was generated using the medaka cyp1a promoter, and this transgenic zebrafish provides a more sensitive biosensor for Ahr activity ( Xu et al., 2015 ). Use of these systems has identified the kidney, liver, and gut as target tissues for TCDD and have also been shown to respond to other dioxin-like chemicals and polyaromatic hydrocarbons ( Xu et al., 2015 ).

4.2.2 Metals

The discovery of elevated lead levels in the drinking water of Flint, Michigan in 2016 has renewed efforts to mitigate the toxic effects of human exposure to metals. In addition to lead, many other metals including copper, platinum, cadmium, and zinc have severe toxic effects on humans and animals. Zebrafish have been used extensively to study the consequences of metal exposure, and a unique transgenic animal has been developed to detect the presence of metals in water. The Tg(mt:egfp) transgenic zebrafish expresses enhanced green fluorescent protein (EGFP) under the transcriptional control of the metal-responsive metallothionein promoter. This line can be used as a reporter for aquatic zinc and cadmium ( Liu, Yan, et al., 2016 ). Recent data had shown that 10 days postfertilization (dpf) zebrafish larvae did not show significant developmental abnormalities even when exposed to levels of heavy metals that exceeded current regulatory limits by 10- or 70-fold for zinc and cadmium, respectively; however, transgene activity was detected following 24 h of exposure to zinc at the current regulatory limit and cadmium at twice the current regulatory limit. Use of the Tg(mt:egfp) zebrafish line provides an advance in the field as it provides a more robust readout for the presence of elevated levels of heavy metals ( Liu, Yan, et al., 2016 ). This is useful, as exposure to a number of different metals is associated with neurodevelopmental deficits. However, further work is needed to refine these tools to respond to additional environmentally relevant metals at a broader range of concentrations.

4.3 Zebrafish Transgenics Shine Light on the Mechanisms of Toxicant-Related Disease

Cellular stress is a central and conserved response to toxin exposure. Many pollutants, including metals, pesticides, and oxidative agents are known or suggested to induce endoplasmic reticulum (ER) stress ( Chen, Melchior, & Guo, 2014 ; Kitamura, 2013 ). Induction of ER stress contributes to a variety of human diseases including neurodegenerative diseases, metabolic dysfunction, inflammatory diseases, and cancer, the risks for which may be compounded by underlying toxicant exposure ( Wang & Kaufman, 2016 ). Oxidative stress is one cause of ER stress, and the metabolism of many toxic compounds, including pesticides and metals, results in the generation of reactive oxygen species. As of yet, there have been few animal models in which to investigate the consequences of toxicant exposure and metabolism, and zebrafish represent a significant advance in this field.

4.3.1 Cellular Stress Reporters

CHOP (also called DDIT3) is a transcription factor that is strongly induced and translocated to the nucleus in response to some types of ER stress ( Harding, Zhang, & Ron, 1999 ; Palam, Baird, & Wek, 2011 ). The transgenic zebrafish line huORFZ contains a GFP transgene under the control of the upstream open reading frame of the human CHOP cDNA ( Lee et al., 2011 ). There are several important features of the huORFZ model that make it ideal for first-line pollution monitoring: (1) exposure to different chemical stressors results in distinct patterns of GFP expression, indicating the cell types and organ systems that respond to a given toxicant; (2) the system is responsive to several pollutants at the range of concentrations enforced by current World Health Organization guidelines; and (3) GFP expression decreased following the exposure period, which suggests that expression is a direct result of the physiological response upon toxicant exposure. Lee et al. (2015) have demonstrated that this transgenic line can be used to detect the presence of environmental contaminants, including heavy metals and EDCs ( Lee et al., 2014 ). The response of this transgenic line is not limited to a particular stressor and can be applied to a range of chemicals or toxicants that induce ER stress.

The heat shock response is a cellular strategy used to protect the cell, by the induction of a number of protein chaperones, which prevents the aggregation of unfolded and misfolded proteins that accumulate due to stress. In addition to heat stress, this survival-promoting response can be induced by aging, protein-folding diseases, and exposure to toxic chemicals ( Scheff Jeremy, Stallings Jonathan, Reifman, & Rakesh, 2015 ). The heatshock promoter driving GFP TgBAC(hspb11:GFP) has been used as a surrogate marker to identify the tissue-specific effects of pesticides ( Shahid et al., 2016 ). This recent study highlights how different cell types are impacted by exposure to the same stressor. Zebrafish embryos were exposed to a number of different pesticides from 9 to 48 hours postfertilization (hpf) and examined for induction of the TgBAC(hspb11:GFP) transgene and muscle integrity. This transgene largely is activated in the muscle and notochord of embryos exposed to pesticides, however, the magnitude of the response varied: azinphosmethyl had a moderate effect on induction of the hspb11 transgene and also only modestly affected muscle integrity, whereas, galanthamine caused severe disruption of muscle integrity and strongly activated the hspb11 promoter ( Shahid et al., 2016 ). Interestingly, the transgene remained active in muscle tissue up to 48 h after the pesticides were removed, indicating the long-lasting effects of toxin exposure on these cells.

4.3.2 Reporters of Endocrine Activity

Activation of estrogen receptors (ERs) is important for developmental processes and sexually dimorphic behaviors. In addition to estradiol and environmental estrogens, several synthetic or exogenous compounds are known to interfere with hormone signaling and have endocrine-disrupting activity (EDCs). One of the main challenges of assessing the effects of EDC is that these compounds are typically functional at very low concentrations and exhibit nonlinear dose responses ( Vandenberg et al., 2012 ). Because of this, the Endocrine Society recommends a “no-threshold” approach to risk assessment for EDC ( Zoeller et al., 2012 ). Zebrafish transgenic reporters thus provide a unique system in which to detect endocrine activity in the absence of gross morphological abnormalities.

Two common zebrafish transgenic reporters that are used for the detection of estrogen receptor signaling are the Tg(5xERE:GFP) ( Gorelick & Halpern, 2011 ; Gorelick et al., 2016 ) and Tg(cyp19a1b:GFP) ( Cano-Nicolau et al., 2016 ; Sonavane et al., 2016 ) zebrafish lines. These lines respond to a range of estrogenic compounds at different doses. For instance, 17α-ethynylestradiol (EE) and diethylstilbestrol (DES) induce GFP expression at the pM to nM range, whereas bisphenol A (BPA) does not induce fluorescence at exposures below the μM range ( Cano-Nicolau et al., 2016 ; Gorelick & Halpern, 2011 ). In addition, reporters of endocrine activity have been used to detect environmental contamination in water samples ( Gorelick et al., 2014 ; Sonavane et al., 2016 ) with similar sensitivity to the established bioluminescent yeast assay ( Gorelick et al., 2014 ). Both of these transgenic lines have been used to detect estrogens in samples collected using the Polar Organic Chemical Integrative Sampler, which concentrates estrogens from environmental water samples. Samples extracted from the membranes are diluted in embryo water, at concentrations higher than that found directly at sampling sites, for exposure and assessment of reporter activity. Due to the nature of the sampling method, these studies determine the estrogenic effects of environmental mixtures. Although use of these transgenic reporters for detection of environmental estrogens has not yet resulted in policy change, these studies highlight the use of zebrafish not only for the detection of estrogenic activity at a single point, but reveal their use in determining variations in contamination levels over time.

While these and other reporters have been highly effective in providing both a practical tool for water quality surveillance and for studying the mechanism of toxin-mediated damage, one major limitation is that by using fluorescent proteins such as GFP, which are slow to mature and have a long half-life, these reporters cannot capture the dynamic response to toxins. A second limitation is that the detection of fluorescent reporters in high-throughput automated imaging systems may be hindered by suboptimal embryo positioning (or nonuniform transgene expression across the zebrafish), such that the brightest parts of some embryos are not imaged accurately. New approaches to surmount these challenges are currently being developed and will enhance the utility of zebrafish transgenics to uncover mechanisms underlying environmental toxicant exposures.

5. TRANSCRIPTIONAL PROFILING TO IDENTIFY CONTAMINANTS

Integration of “-omic” technologies into environmental toxicology has been occurring at a rapid pace as new advances in image processing and data analysis make the use of these applications more feasible. Connectivity mapping is a data-driven approach combining transcriptomics and machine learning technology, and has previously been used to link disease and drug-induced phenotypes on the basis of differential gene expression patterns ( Lamb et al., 2006 ). This approach has been applied to assess exposure and toxicity to chemical groups based on mechanism(s) of action and transcriptomic changes. Software packages have been developed to allow users to compare gene expression profiles under their treatment or exposure conditions to those archived in publicly available databases ( Sandmann, Kummerfeld, Gentleman, & Bourgon, 2014 ). Wang et al. (2016) published the first use of connectivity mapping in environmental health using zebrafish. By mining publicly available microarray datasets, the group compared the transcriptional responses to a range of chemical exposures and doses in different organs from zebrafish and fathead minnow. Mapping is more successful within species and among those samples run on the same platform. As the cost of mRNA sequencing technologies reduces, whole genome data will be available for an increasing number of model systems under different experimental conditions. Connectivity mapping also offers researchers the opportunity to generate hypotheses about the mechanisms of action for environmental pollutants or toxins for which mechanistic pathways were previously unknown.

Many toxins specifically affect the liver, as this is the primary site of xenobiotic metabolism in vertebrates. Pathologies ranging from necrosis and fatty liver, to steatohepatitis and liver cancer have been found to result from occupational and environmental exposure to chemicals and toxicants ( Al-Eryani et al., 2015 ; Wahlang et al., 2013 ). Toxin-specific hepatic responses have been identified in zebrafish using gene expression analysis on a variety of platforms to determine the hepatic response to a range of toxins, including arsenic, acetaminophen, and ethanol ( Xu, Lam, Shen, & Gong, 2013 ; Zhang, Li, & Gong, 2014 ). A well-defined genome, easy access to target organs, and conserved responses to toxins make zebrafish amenable to emerging genomic, proteomic, and metabolomics approaches to better understand the molecular changes caused by toxins.

6. HIGH-THROUGHPUT SCREENING FOR TOXICITY STUDIES

A major goal for toxicology studies is to be able to screen many compounds in a short amount of time and with accuracy in predicting human toxicity. In 2007, the EPA began the ToxCast program to screen chemicals in order to develop protocols that would lead to improved human toxicity prediction ( Dix et al., 2007 ). The pilot study, ToxCast Phase I, included 310 compounds (mostly pesticides) that were screened in a large number of medium- and high-throughput screening assays ( Judson et al., 2010 ). That same year, the National Research Council published a report titled “Toxicity testing in the 21st Century: A Vision and Strategy” ( National Research Council, 2007 ), which prompted rapid expansion of ToxCast, and in Phase II of the ToxCast program, the chemical library was expanded to 1878 compounds for which testing concluded in 2013 ( Richard et al., 2016 ). ToxCast Phase I and II library compounds have been tested in model organisms including C. elegans and zebrafish ( Boyd et al., 2016 ; Padilla et al., 2012 ; Sipes, Padilla, & Knudsen, 2011 ). Phase III of the ToxCast program contains greater than 3800 unique chemicals and compounds under evaluation ( Richard et al., 2016 ).

Toxicology in the 21st Century (Tox21) program is a collaboration between the NIEHS National Toxicology Program, the EPA, and the National Center for Advancing Translational Science to test more than 10,000 environmental chemicals and drugs to elucidate their toxicity in biochemical and cell-based assays ( Collins, Gray, & Bucher, 2008 ). The FDA’s ToxCast joined the collaboration in 2010 and are now jointly referred to as the “ToxCast chemical library” ( Richard et al., 2016 ). The European community has also responded with the EU R egistration, E valuation, A uthorization and restriction of CH emical substances (REACH) legislation, requiring the collection of toxicity data for chemicals that are produced or marketed in quantities in excess of one ton per year ( Selderslaghs, Blust, & Witters, 2012 ). Several research centers in Europe now use zebrafish as the central animal model for toxicology studies and centralized groups have issued a white paper calling for increased resources for using zebrafish for toxicology research ( www.eufishbiomed.kit.edu ).

Limitations to these approaches are that xenobiotic metabolism cannot be studied in vitro, determining active in vivo doses and blood concentrations from in vitro studies is not possible, understanding the effects of chronic exposure is impossible in vitro, and knowing whether or when a given genetic or signaling perturbation would result in a phenotypic change in an animal is difficult to ascertain ( Tice, Austin, Kavlock, & Bucher, 2013 ).

Zebrafish are being used as a first-pass screen to identify chemicals with the highest likelihood of posing risk to humans and require further testing ( Dix et al., 2007 ). Researchers at the EPA used the zebrafish developmental assay to add information to the toxicity assay database of the ToxCast Phase I library ( Padilla et al., 2012 ). In this first large-scale screen of the effects of environmental contaminants zebrafish, embryos were exposed from 6 hpf to 5 dpf to a single dose and then a concentration range from 1 nM to 80 μM. Survival and morphological defects were assessed at 6 dpf. This was expanded in a subsequent study that analyzed the effects of several hundred chemicals from the ToxCast Phase II library on 18 different endpoints in zebrafish larvae at 5 dpf ( Truong et al., 2014 ). More recently, 1060 compounds from the ToxCast I and II chemical libraries have been tested in a phenotype-based screen in zebrafish to predict teratogenic effects. This study showed that hypoactivity at 24 hpf in exposed zebrafish embryos is associated with an increased risk of 17 specific developmental abnormalities as assessed 5 dpf larvae ( Reif et al., 2016 ). Interestingly, this study also identified a group of chemical compounds that caused the same degree of hypoactivity at 24 hpf, with no corresponding morphologic defect at 5 dpf, indicating that this protocol may prevent false negatives. Efforts to build databases and develop assays to predict human toxicity have capitalized on the use of zebrafish as a quick, medium throughput in vivo system to accurately predict human toxicity.

In 2009, an international group of pharmaceutical companies formed a consortium to develop a zebrafish development assay that could correctly classify a set of 10 teratogenic and 10 nonteratogenic compounds ( Gustafson et al., 2012 ). The results of these toxicity tests were compared to mammalian data, and found to have an overall concordance of 60–70%. In a second phase of this consortium project, 38 proprietary pharmaceutical compounds were tested by two independent laboratories, and 79% of the classifications were the same between the laboratories, although the laboratories differed in their concordance with in vivo data ( Ball et al., 2014 ). The Dechorionated Zebrafish Embryo Developmental toxicity assay was developed to identify the no-adverse-effect-level (NOAEL) and the concentration resulting in 25% lethality (LC 25 ) for a training set of 31 compounds ( Brannen, Panzica-Kelly, Danberry, & Augustine-Rauch, 2010 ). This approach yielded 87% concordance with published in vitro teratogenicity data ( Brannen et al., 2010 ). Improvements to this assay, including enzymatic removal of the chorion, repeating the assay with a distinct set of text compounds, and using various zebrafish strains, have been attempted to make a direct comparison between the chorion-on data published by the pharmaceutical company consortium and the chorion-off data to determine whether the presence of the chorion affected the sensitivity and specificity of the zebrafish embryo assay ( Ball et al., 2014 ; Brannen et al., 2010 ; Gustafson et al., 2012 ; Panzica-Kelly, Zhang, & Augustine-Rauch, 2015 ).

New advances in the morphological assessment of toxicant-exposed zebrafish larvae allow for the determination of the effects of test compounds on developmental endpoints. Computational approaches, including the Cellomics ® ArrayScan ® V TI high-content image analysis platform reduce the time required for analysis and reduce variability between experiments while providing an image that can be kept for permanent record or reevaluated manually ( Deal et al., 2016 ). Bright field image analysis will identify a large number of phenotypes that may be undetectable using other methods. The use of zebrafish offers a valuable tool for high-throughput screening of compounds with demonstrated accuracy in predicting human toxicity. Further, development of these technologies and platforms will be important for identifying target organs and generating hypotheses about mechanisms of action for chemicals for which no biological data are available.

7. ASSESSING HEALTH IMPACTS OF ENVIRONMENTAL EXPOSURES USING ZEBRAFISH

The ability to assess tissues for toxin accumulation and its associated phenotypes can lead to valuable insights into disease processes and enable therapeutic compound screening. While transgenic reporter lines can monitor differentiation of distinct cell lineages and detect the induction of signaling pathways, much of the data acquisition is limited to low- and medium-throughput applications due to the time required for screening individual zebrafish embryos and larvae by fluorescence microscopy.

7.1 Automated Reporter Quantification In Vivo (ARQiv)

Automated reporter quantification in vivo (ARQiv) is a high-throughput screening platform that uses a microplate reader to detect changes in the intensity of transgenic fluorescent reporters in live zebrafish embryos and larvae over time ( Walker et al., 2012 ). Recently, ARQiv technology has been applied to test FDA-approved drugs and their ability to increase the number of insulin-producing pancreatic β cells in a transgenic reporter zebrafish line ( Tg(ins:PhiYFP-2a-nsfB; sst2:tagRFP)lmc01 ), demonstrating the feasibility of this approach for both quantification of cell number and fluorescent reporter intensity ( Wang et al., 2015 ). An increase of as little as 10 β cells in the developing pancreas was detected, highlighting the ability of this technique to identify small changes in the development of this important organ.

Although transgenic reporters are routinely used to visualize the effects of drugs and chemical compounds on developing organ systems ( Lam et al., 2011 ; Ma et al., 2015 ), this technology can also be applied to environmental exposures using the same tissue- and signaling pathway-specific transgenic reporter lines to assess toxin-induced effects on the development of cell types and organ structures. One major limitation is that it only provides quantification of reporter levels without corresponding images to allow for the analysis of morphological changes associated with changes in reporter activity ( Wang et al., 2015 ). Data generated using ARQiv will need to be coupled with that from other imaging techniques to obtain full understanding about the phenotypic effects of a particular exposure.

7.2 Laser Ablation-Inductively Coupled Plasma-Mass Spectroscopy (LA-ICP-MS)

Laser ablation-inductively coupled plasma-mass spectroscopy (LA-ICP-MS) can be used to provide spatial information about element distribution in biological samples ( Hare, Austin, & Doble, 2012 ). This technique can be used in calcified tissue (teeth) and soft tissue (placenta) ( Arora et al., 2014 ; Niedzwiecki et al., 2016 ). We are currently optimizing the use of LA-ICP-MS to determine tissue accumulation and organ-specific distribution of elements in whole zebrafish larvae (data not shown). The technique is able to detect compounds at concentrations below parts-per-million and has spatial resolution capacity at the micrometer range allowing for detailed analysis of tissue; however, quantification of trace elements within tissue samples using LA-ICP-MS analysis is not yet reliable due to properties of the ablation process ( Hare et al., 2012 ).

7.3 Automated Assessment of Behavior and Morphologic Phenotypes

Complex developmental effects associated with exposures can be studied in zebrafish using behavioral profiling ( Rihel et al., 2010 ) and phenotype-driven screens ( Gallardo et al., 2015 ). Behavioral profiling is most useful for modeling effects on brain activity and has recently been used to identify phenotypic suppressors of autism in a zebrafish genetic model of hyperactivity ( Hoffman Ellen et al., 2016 ). Similar efforts could be used to identify environmental modifiers of genes associated with autism spectrum and other neurological disorders. Phenotype-driven chemical screening has been used to identify compounds that altered the collective migration of fluorescently marked cells ( Gallardo et al., 2015 ).

Automation of image capture and phenotype analysis will improve the ability of researchers to screen larger libraries of compounds over wider concentration ranges, while limiting bias in the assays ( Deal et al., 2016 ; Jeanray et al., 2015 ; Mikut et al., 2013 ). Optimized techniques for embryo immobilization will enable imaging the developing zebrafish larvae using state of the art techniques including light sheet fluorescence microscopy ( Höckendorf, Thumberger, Wittbrodt, 2012 ; Kaufmann, Mickoleit, Weber, & Huisken, 2012 ). Zebrafish can facilitate analysis of developmental and structural changes over time, but require development of advanced video capabilities. Recently, an open source application for the video analysis of movement of larval zebrafish has been created for academic use ( Cario, Farrell, Milanese, & Burton, 2011 ). The rapid advances in imaging and computational technologies to identify the morphologic consequences of toxicant exposure in zebrafish, put this model at the forefront of the field with the potential to advance the identification of teratogenic and tissue-specific effects of toxins.

8. LONG-TERM AND TRANSGENERATIONAL EFFECTS OF TOXIN EXPOSURES

Exposure to environmental toxicants during development can have both acute consequences to the embryo, leading to congenital anomalies and poor birth outcomes, as well as long-term health consequences throughout the life of an individual. In addition, exposure to low doses of environmental contaminants can have latent health effects that are not apparent for years, even after the cessation of exposure. The fetal origins hypothesis, also called Barker hypothesis, was first described by David Barker in 1986 following the observation that poor infant nutrition was associated with poor cardiovascular outcomes among men in England and Wales ( Barker & Osmond, 1986 ). Adverse health effects in this cohort are thought to be the result of altered developmental programming or physiological changes that make an individual susceptible to disease.

A well-known examples of this phenomenon is the causal association between the development of vaginal clear cell adenocarcinoma in women who were exposed to diethylstilbestrol in utero ( Hatch, Palmer, Titus-Ernstoff, et al., 1998 ; Herbst, Ulfelder, & Poskanzer, 1971 ). This hypothesis has recently been tested in zebrafish to study the latent effects of embryonic exposure to atrazine, an herbicide and suspected endocrine disruptor ( Wirbisky et al., 2015 , 2016 ), and TCDD, a persistent environmental pollutant ( Baker, Peterson, & Heideman, 2013 ). Both male and female adult zebrafish that developed from embyros exposed to atrazine, a widely used herbicide, demonstrate altered expression of genes related to neuroendocrine function ( Wirbisky et al., 2016 , 2015 ). In another study, early life exposure to low doses of TCDD during the embryonic period caused few malformations in the fish during the exposure period; more profound were the transgenerational effects of early exposure: the offspring of adults which developed from embryos exposed to TCDD showed morphological abnormalities, including skeletal defects, and reduced reproductive success ( Baker et al., 2013 ). Zebrafish that were exposed to TCDD during the sex determination period (3–7 weeks postfertilization) displayed skeletal anomalies in adulthood and mismatches between secondary sex characteristics and the sex of the gonads as determined by histological analysis ( Baker et al., 2013 ). These studies exemplify the power of the zebrafish system to feasibly demonstrate early, late, and transgenerational effects of toxin exposure.

9. PATHWAYS AND MECHANISMS OF TOXICANT-INDUCED DISEASE

Zebrafish can provide a powerful tool to investigate the mechanisms of action of environmental pollutants and its related diseases, and can be used to test therapeutic candidates or intervention measures to mitigate the effects of environmental contaminants, with the goal of translation to human disease. Here, we highlight examples of mechanistic insights generated from zebrafish models of exposure, including inorganic arsenic, BPA and TCDD ( Fig. 2 ).

Using zebrafish to model the health effects of toxicant exposure. Zebrafish exposed to a range of water-soluble environmentally relevant contaminants during different stages of the life cycle display a wide range of outcomes, from reduced reproductive success to skeletal and neurodevelopmental defects. BPA , bisphenol A; TCDD , 2,3,7,8-tetrachlorodibenzo- p -dioxin. Illustrations by Christopher Smith. Copyright Mount Sinai Health System 2016. Images used with permission.

9.1 Arsenic

Inorganic arsenic is a naturally occurring element that epidemiological studies have linked with multiple adverse health outcomes ( Vahter, 2008 ). Arsenic is classified as a human carcinogen by the International Agency for Research on Cancer and long-term exposure is associated with increased risk of several cancers, including bladder, kidney, liver, and skin cancer ( IARC, 2004 ; Wang et al., 2014 ). Data from the first longitudinal study of people chronically exposed to inorganic arsenic through drinking water has found that the latency for health effects can be decades ( Ahsan et al., 2006 ; Argos et al., 2010 ). Studying the underlying mechanisms of arsenic toxicity in zebrafish can provide much needed information of how arsenic causes disease in humans.

Although arsenic is one of the most common metalloid contaminants of drinking water, and can be found at high levels in common foods such as rice and apple juice ( Davis et al., 2012 ; Sauvé, 2014 ), the precise mechanism of arsenic toxicity is relatively unknown. Work in tissue culture cells and rodent models has identified oxidative stress, biotransformation and methylation, and ER stress as potential mechanisms of arsenic-induced toxicity ( Gamble et al., 2005 ; Hughes, 2002 ; Vahter, 2002 ), although studies linking aberrant cellular processes to specific arsenic-induced disease phenotypes are lacking. Work in zebrafish is shedding light on this field.

Zebrafish express aquaglyceroporins and the trivalent arsenic specific methyltransferase ( zas3mt ), enzymes required for the uptake and metabolism of inorganic arsenic, respectively ( Hamdi et al., 2009 , 2012 ). Early studies of zebrafish models of inorganic arsenic exposure provided descriptive analysis of the effects of arsenic on the gross morphological development of zebrafish embryos and larvae, demonstrating acute toxicity and cardiovascular defects ( Li et al., 2009 ; Seok et al., 2007 ). While information about the rate of arsenic metabolism by zebrafish in vivo and the production of specific metabolites are still emerging, it is clear that rodent models do not always accurately reflect the effects of human arsenic exposure. For instance, studies in rodents showed increased excretion and slower but more extensive methylation of arsenic when compared to humans so that ingested arsenic remains in the rodent blood stream for prolonged periods ( Hallauer et al., 2016 ; States et al., 2011 ).

Zebrafish embryos treated with inorganic arsenic have multiple defects, and some studies implicated downregulation of Dvr1, a factor involved in mesoderm induction and the establishment of left-right asymmetry ( Li et al., 2012 ). These processes are essential for proper cardiac morphogenesis. Interestingly, depletion of Dvr1 using morpholino knockdown led to heart defects that were similar to those seen upon exposure to 2 mM inorganic arsenic from 4 to 48 hpf ( Li et al., 2012 ). Overexpression of human GDF1, a homolog of Dvr1, led to a reduction in the number of zebrafish that displayed morphological defects upon arsenic exposure ( Li et al., 2012 ). Further studies from the same group showed folic acid prevented arsenic-induced toxicity in zebrafish by reducing generation of reactive oxygen species and rescuing the decrease in Dvr1 expression ( Ma et al., 2015 ). However, the protective effect of folic acid diminished after 48 hpf ( Ma et al., 2015 ), indicating that other mechanisms may underlie later developmental anomalies caused by arsenic exposure.

Transcriptomic and metabolomic approaches in zebrafish have also been used to gain insight into the molecular mechanisms of arsenic toxicity, particularly in the adult liver ( Lam et al., 2006 ; Li et al., 2016 ; Xu et al., 2013 ; Yang et al., 2007 ). The effect of acute arsenic exposure and changes of gene expression patterns over time in the adult liver was first examined using microarray, with gene expression changes occurring as early as 8 h of exposure ( Lam et al., 2006 ). Differentially expressed genes were grouped into categories to examine the adaptive response of the zebrafish liver to arsenic exposure, and revealed that arsenic-induced liver injury is the result of DNA and protein damage and oxidative stress resulting from the metabolism of inorganic arsenic. Gene ontology and pathway analysis of RNA-SAGE data were applied and used to identify a panel of biomarker genes to predict arsenic toxicity ( Xu et al., 2013 ). Network analysis identified nr2f2 , jun , k-ras , and apoE as four central factors that were upregulated in zebrafish liver following arsenic exposure. Each of these factors is implicated in pathways that can contribute to arsenic-induced liver disease: Jun and Kras are known oncoproteins, Nr2f2 regulates many genes involved in oxidative stress, and drug metabolism and ApoE is required for lipoprotein synthesis. Interestingly, arsenic exposure has been shown to accelerate the formation of atherosclerosis in ApoE deficient mice, highlighting the conservation of pathways affected by arsenic exposure in zebrafish and mammalian models ( States et al., 2012 ; States, Srivastava, Sen, & D’Souza, 2007 ).

Chronic exposure of zebrafish to environmentally relevant concentrations revealed retention of arsenic in the eye, skin, and liver of 6-month-old fish and resulted in increased heart rate during larval stages and neurologic defects ( Hallauer et al., 2016 ). Progeny of arsenic-exposed fish had reduced biomass at 3 months of age relative to the progeny of their unexposed siblings ( Hallauer et al., 2016 ). Zebrafish studies have recapitulated the effects of arsenic on the cardiovascular system ( Hallauer et al., 2016 ; Li et al., 2012 ) and have shown alterations in liver metabolism and liver function ( Lam et al., 2006 ; Li et al., 2016 ; Xu et al., 2013 ). Our research is focused on using zebrafish to understand the mechanisms that underlie arsenic-induced liver disease in human populations ( Mazumder, 2005 ; Santra, Das Gupta, De, Roy, & Guha Mazumder, 1999 ). Metabolic changes in the liver of adult zebrafish after acute arsenic exposure was investigated using gas chromatography coupled with mass spectroscopy ( Li et al., 2016 ), identifying 34 potential metabolite markers of arsenic exposure. Additionally, histological examination of the livers of arsenic-exposed zebrafish showed cellular changes and accumulation of lipid droplets, liver function tests showed little alteration ( Li et al., 2016 ), suggesting that metabolic changes may be a sensitive method to detect alterations in liver function induced by arsenic. Although the use of zebrafish to study arsenic toxicity is relatively recent, this model system has provided important insights into both the acute ( Li et al., 2016 ; Xu et al., 2013 ) and chronic effects of arsenic exposure ( Hallauer et al., 2016 ). Zebrafish studies of the effects of arsenic exposure will provide insight into the mechanisms of these physiological consequences and will also allow for the examination of transgenerational effects more feasibly than with rodent models.

9.2 Bisphenol A

BPA is one of the most common endocrine-disrupting environmental contaminants. It is a high production volume chemical and is present in many consumer and industrial products such as plastics. It is also present in the environment as a result of manufacturing processes and leaching from the products in which it is used. BPA is defined as an endocrine disruptor because of its ability to elicit both proestrogenic and antiestrogenic effects by binding to estrogen receptors ERα and ERβ and altering transcription in tissue- and context-specific manners ( Santangeli et al., 2016 ). BPA binds to the zebrafish estrogen-related receptor gamma (ERRγ) in vivo ( Tohmé et al., 2014 ). BPA has been reported to have adverse effects on reproductive health, early development, and contributes to obesity ( Rochester, 2013 ). Both mammalian and zebrafish exposure studies have revealed related phenotypes.

Environmentally relevant doses of BPA were found to inhibit oocyte maturation by binding to the membrane estrogen receptor, Gper, and activating Egfr/Mapk3/1 signaling, which prevents resumption of meiosis ( Fitzgerald, Peyton, Dong, & Thomas, 2015 ). Interestingly, this pathway is independent of signaling through the estrogen receptor ( Fitzgerald et al., 2015 ). BPA is suggested to act through epigenetic mechanisms through histone modification and alteration of DNA methylation ( Faulk et al., 2016 ; Kundakovic & Champagne, 2011 ; Santangeli et al., 2016 ), and zebrafish studies have provided key mechanistic insights. The effect of BPA on histone methylation patterns and DNA methylation has recently been shown in adult zebrafish ( Laing et al., 2016 ; Santangeli et al., 2016 ). Global DNA methylation has also recently been shown to be reduced in the ovaries and testes of adult zebrafish exposed to 15 μg/L BPA for 7 days ( Liu, Zhang, et al., 2016 ) and 1 mg/L BPA for 15 days ( Laing et al., 2016 ). Adult female zebrafish exposed to environmentally relevant concentrations of BPA, from 5 to 20 μg/L, displayed nonmonotonic effects in that the lowest dose tested led to a complete block in ovulation, accompanied by more significant reduction in gene expression of the estrogen receptors esr1 and esr2a , and induction of apoptosis markers caspase3 and p53 ( Santangeli et al., 2016 ). Expression of the DNA methyltransferases dnmt1 and dnmt3 were upregulated in the ovaries of female zebrafish exposed to 5 μg/L BPA. Some of these gene expression changes were associated with changes in the levels of H3K4me3 and H3K27me3 levels ( Santangeli et al., 2016 ). In contrast, adult male and female zebrafish exposed to higher doses of BPA (up to 1 mg/L) were shown to have reduced expression of dnmt1 in the liver and ovaries when exposed to 10 μg/L, 100 μg/L, and 1 mg/L BPA, while no significant differences in expression level were observed in the testes of male zebrafish at the same exposure concentrations ( Laing et al., 2016 ). Interestingly, the DNA methylation patterns were not strictly correlated with changes in gene expression. For instance, while no change in dnmt1 expression was observed in the testes, analysis of 11 CpG sites in the dnmt1 promoter revealed significant increases in some of the sites in this tissue. In the ovary, where the most consistent changes in dnmt1 expression were observed, no significant changes in site-specific DNA methylation in the dnmt1 promoter were found ( Laing et al., 2016 ).

In addition to studying BPA-induced defects in reproduction, zebrafish have also been used to understand the neurotoxic effects of BPA ( Cano-Nicolau et al., 2016 ; Kinch, Ibhazehiebo, Jeong, Habibi, & Kurrasch, 2015 ; Saili et al., 2012 ). In the zebrafish brain, BPA can activate gene expression through the canonical estrogen receptor signaling pathway ( Chung, Genco, Megrelis, & Ruderman, 2011 ). Zebrafish exposed to BPA for a narrow (8–58 hpf) or longer (8–120 hpf) window were examined for effects on behavior. Zebrafish larvae (5 dpf) that were exposed to low dose BPA from 8 to 58 hpf demonstrated hyperactivity, and adult zebrafish that were exposed to low dose BPA from 8 to 120 hpf had behavioral and learning deficits, including larval hyperactivity and reduced ability to choose the correct arm of a T-maze to avoid an electric shock, compared to unexposed controls ( Saili et al., 2012 ).

Using transgenic Tg(cyp19a1b:GFP) reporter fish, a reporter of estrogen signaling, zebrafish larvae exposed to BPA on 4 or 7 dpf resulted in activation of the estrogen-specific marker which likely occurred through activation of ERα ( Cano-Nicolau et al., 2016 ). Reporter expression was localized to specific brain regions including the posterior telencephalon, preoptic area, and caudal hypothalamus. In the zebrafish brain, it has also recently been shown that exposure of developing zebrafish to low doses BPA caused precocious neurogenesis in the hypothalamus which resulted in hyperactivity and brain changes ( Kinch et al., 2015 ). Together, these data show that zebrafish are capable of demonstrating not only the molecular and cellular responses to the endocrine disruptor BPA, but also provide evidence of the pathological effects of BPA exposure.

Most recently, zebrafish have been used to study the toxic effects of BPA and the products of its degradation ( Makarova, Siudem, Zawada, & Kurkowiak, 2016 ). The degradation products of BPA were found to have lower binding affinity for both human and zebrafish estrogen receptors than BPA itself but one degradation product, 4-isopopylphenol, was predicted to have a higher binding affinity for the human ERRγ and slightly lower affinity for zebrafish ERRγA. 4-Isopopylphenol has the ability to permeate biological membranes similar to BPA, but appears to be more toxic as it caused acute lethality to zebrafish embryos while the same dose of BPA did not. These zebrafish studies emphasize that degradation products of environmental contaminants can be more toxic than their parent compounds, and that toxicity testing of intermediates may be warranted ( Gamse & Gorelick, 2016 ).

Zebrafish have provided useful insights into the effects of BPA on the developing brain and reproductive organs. These systems are most the most likely to be affected by environmental exposures to BPA and similar compounds that rely heavily on estrogen signaling ( Patisaul & Adewale, 2009 ; Saili et al., 2012 ). Studies of BPA toxicity in zebrafish have highlighted the nonmonotonic effects of this common environmental contaminant and other EDCs ( Santangeli et al., 2016 ; Vandenberg et al., 2012 ).

TCDD is one of the most widely studied environmental contaminants in zebrafish ( Carney, Prasch, Heideman, & Peterson, 2006 ). This chemical is a polychlorinated dibenzo- p -dioxin, an anthropogenic, lipophilic persistent environmental contaminant, commonly found in air and soil as the result of solid waste incineration and industrial processing. Human occupational and environmental exposure may be associated with a wide range of chronic diseases, including cancer, diabetes, endometriosis, cardiovascular disease, reduced testosterone, and thyroid hormone levels ( White & Birnbaum, 2009 ). The effects of aromatic hydrocarbon exposure on the health of wild fish populations are more difficult to assess; however, lake trout populations in regions with high levels of aromatic hydrocarbon contamination have been unable to sustain their numbers ( King-Heiden et al., 2012 ). Zebrafish have been proposed as a model to understand not only the health effects of human exposure to aromatic hydrocarbons and dioxin-like compounds, but also to predict the effects of contamination on wild fish populations. A comprehensive review of the contributions of the zebrafish model to our understanding of the molecular mechanisms of TCDD reproductive and developmental toxicity has been published elsewhere ( King-Heiden et al., 2012 ).

Zebrafish have been used to study TCDD-induced endocrine disruption and reproductive toxicity ( Baker et al., 2013 ; Heiden et al., 2008 ), cardiovascular toxicity ( Antkiewicz, Burns, Carney, Peterson, & Heideman, 2005 ; Goldstone & Stegeman, 2006 ), and skeletal abnormalities ( Baker et al., 2013 ; Burns, Peterson, & Heideman, 2015 ; Henry, Spitsbergen, Hornung, Abnet, & Peterson, 1997 ; Teraoka et al., 2006 ). Developmental malformations in zebrafish embryos and larvae exposed to TCDD are prevented by depletion of Ahr2 ( Prasch et al., 2003 ), indicating that metabolism is required for TCDD toxicity. Cyp1a transgenic reporter zebrafish have been used to investigate the mechanisms of TCDD toxicity and to identify target organs for the effects of TCDD ( Kim et al., 2013 ; Mattingly, McLachlan, & Toscano, 2001 ; Xu et al., 2015 ); however, pathways independent of Cyp1a1 also contribute to the developmental toxicity of TCDD as knockdown of zebrafish cyp1a does not prevent TCDD-induced phenotypes ( Carney, Peterson, & Heideman, 2004 ). Here, we compile some of the most recent studies of zebrafish exposed to TCDD.

TCDD has been shown to affect both ovarian function and follicle maturation ( Baker, Peterson, & Heideman, 2014 ; Heiden et al., 2008 ). Studies in numerous fish species have demonstrated many impairments in female reproduction that are caused by TCDD ( King-Heiden et al., 2012 ). A microarray analysis of the zebrafish adult ovary examined the transcriptional changes that precede the physiologic dysfunction following exposure to a TCDD dose curve ( Heiden et al., 2008 ). Exposure to TCDD resulted in downregulation of genes involved in estradiol synthesis and follicle maturation, as well as genes encoding structural proteins Krt4 and Lgals3l ( Heiden et al., 2008 ). While this study found that gene expression changes were not dose dependent and that a majority of the differentially expressed transcripts were unknown or poorly characterized, ~40% of the differentially expressed probes contained both putative aryl hydrocarbon-response elements and estrogen response elements.

Cardiac toxicity is one of the most obvious end points of zebrafish exposure to TCDD ( King-Heiden et al., 2012 ). Gene expression analysis over a time course was performed to understand the molecular pathways that are altered in response to TCDD exposure ( Carney, Chen, et al., 2006 ). Within 1 h of exposure, a cluster of 42 genes involved in xenobiotic metabolism, proliferation, contractility, and regulation of heart development were induced ( Carney, Chen, et al., 2006 ). In addition a “cell cycle gene cluster” was downregulated in zebrafish exposed to TCDD and negative regulators of cell cycle progression were upregulated, indicating that reduced cardiomyocyte number may underlie TCDD-induced cardiac toxicity ( Carney, Chen, et al., 2006 ). Increased and ectopic expression of Bmp4 and Notch1b transcripts in the region of nascent cardiac valve formation were found to be responsible for TCDD-induced failure of heart valve formation in the zebrafish ( Mehta, Peterson, & Heideman, 2008 ). Failure to restrict these transcripts, as determined by in situ hybridization, was associated with loss of endothelial cell pattern in the region where this morphogenic process should occur. This study highlights one of the most significant advantages to using the zebrafish system, in that alterations in stereotypical developmental processes can yield insight into the cellular and molecular mechanisms underlying toxicity.

Skeletal malformation is another predominant developmental defect associated with TCDD exposure in fish and rodent species ( Baker et al., 2014 ; Birnbaum, Harris, Stocking, Clark, & Morrissey, 1989 ; Henry et al., 1997 ; King-Heiden et al., 2012 ). Craniofacial malformations in TCDD-exposed zebrafish are dependent on Ahr2/Arnt1 signaling ( Prasch, Tanguay, Mehta, Heideman, & Peterson, 2006 ; Prasch et al., 2003 ). A transgenic reporter Tg(sox9b:EGFP) , which marks perichondrial endoderm in the developing jaw, was used to demonstrate that craniofacial abnormalities in TCDD-exposed zebrafish larvae resulted from reductions in chondrocyte size and number and decreases in ossification of the jaw ( Burns et al., 2015 ). In addition to being a marker of craniofacial and jaw development in the zebrafish, sox9b is required for this process. Heterozygous sox9b mutant zebrafish are more susceptible to TCDD-induced craniofacial malformations, and overexpression of sox9b in TCDD-treated zebrafish mitigated the effects of the toxicant on jaw development ( Xiong, Peterson, & Heideman, 2008 ). Scoliosis is frequently observed in adult fish following exposure to TCDD during the embryonic or larval periods ( Baker et al., 2013 , 2014 ).

Zebrafish research into the pathways and molecular mechanisms underlying TCDD toxicity have provided information about outcomes relevant to human populations, most notably cardiac and reproductive defects. TCDD is also the most commonly studied environmental toxicant with regard to transgenerational effects, as discussed in Section 8 ( Baker, King-Heiden, Peterson, & Heideman, 2014 ; Baker et al., 2014 ).

10. LIMITATIONS TO THE ZEBRAFISH MODEL SYSTEM

While zebrafish will allow researchers to answer many questions that are limited by the realities of epidemiological researchers, there are several limitations to this model. For instance, the physiological differences between zebrafish and mammals mean that disease outcomes such as asthma or placental defects are not observable in zebrafish. However, although not all disease-related phenotypes can be identified in zebrafish, many of the developmental and signaling pathways leading to these diseases are conserved between zebrafish and humans ( Padilla et al., 2012 ).

A second consideration is that in zebrafish, some exposures may not be equivalent to the experience of human populations. In most studies, the toxicant is added directly to the water, recapitulating a dermal exposure during the early stages of zebrafish development when the embryos are not swallowing water in order to breathe. However, many toxicants are introduced into the human body via oral exposure through contaminated drinking water or food, and, as such exposure is intermittent and affects involves the gastrointestinal system. This may lead to substantial differences in the absorption, tissue distribution, metabolism, and excretion depending on the uptake and biotransformation pathways based on the route of exposure. Metabolic differences between zebrafish and mammals may also be affected by differences in the expression patterns of xenobiotic metabolism enzymes and incompletely conserved enzyme functions ( Saad et al., 2016 ), which may also contribute to the differences in dosing required to elicit phenotypes. Urinary biomarkers of exposure and metabolism are also unavailable from zebrafish.

Studies have demonstrated that gender can play an influential role in response to toxin exposure. For example, arsenic exposure in humans leads to changes in DNA methylation in isolated cord blood cells that are different in males and females ( Pilsner et al., 2012 ), and endocrine disruptors have been shown to have different neurobehavioral effects in boys and girls ( Evans et al., 2014 ; Roen et al., 2015 ). While zebrafish have no discernible sex chromosomes and do not become sexually dimorphic until 3 weeks postfertilization ( Sola & Gornung, 2001 ; Tong, Hsu, & Chung, 2010 ), toxicant exposure during this window can influence sex characteristics as seen with early TCDD exposure ( Fig. 2 ) ( Baker et al., 2013 ).

11. CONCLUSIONS

As manufacturing processes and development of new synthetic compounds proceed in order to keep pace with the growing world economy, environmental health, and the effects of toxicant exposure are emerging as critical areas of research. The main benefit to using zebrafish in toxicology and environmental health studies is that their unique combination of developmental features provides a system with the benefits of both in vitro and in vivo schemes. Combining the large scale of embryo production with rapid development allows for short-term assessment of toxicity in a whole animal system. In addition, the relative ease and comparatively low cost of raising large numbers of individuals allows for unprecedented investigation into latent effects and adverse outcomes in response to early life exposure to environmental contaminants. Many of the genetic, molecular, and cellular processes are conserved between zebrafish and mammals, allowing close applicability to human exposure and disease. As such, studies using zebrafish have uncovered important insights into the effects of environmental contaminants on normal development in a live vertebrate system.

Acknowledgments

The authors would like to acknowledge Drs. Kirsten C. Sadler and Anjana Ramdas for critical reading of this chapter. Our research is supported by T32HD049311-01A1 (K.B.) and K08DK101340 and the Mindich Child Health and Development Institute at Mount Sinai (J.C.).

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